Apparatus for fluid line management in a bioprocessing system

ABSTRACT

In an embodiment, a tubing module for a bioprocessing system includes a first tubing holder block configured to receive at least one pump tube and hold the at least one pump tube in position for selective engagement with a peristaltic pump, a second tubing holder block configured to receive a plurality of pinch valve tubes and hold each pinch valve tube of the plurality of pinch valve tubes in position for selective engagement with a respective actuator of a pinch valve array, and wherein the first tubing holder block and the second tubing holder block are interconnected.

BACKGROUND Technical Field

Embodiments of the invention relate generally to bioprocessing systemsand methods and, more particularly, to a bioprocessing system andmethods for the production of cellular immunotherapies.

Discussion of Art

Various medical therapies involve the extraction, culture and expansionof cells for use in downstream therapeutic processes. For example,chimeric antigen receptor (CAR) T cell therapy is a cellular therapythat redirects a patient's T cells to specifically target and destroytumor cells. The basic principle of CAR-T cell design involvesrecombinant receptors that combine antigen-binding and T-cell activatingfunctions. The general premise of CAR-T cells is to artificiallygenerate T-cells targeted to markers found on cancer cells. Scientistscan remove T-cells from a person, genetically alter them, and put themback into the patient for them to attack the cancer cells. CAR-T cellscan be derived from either a patient's own blood (autologous) or derivedfrom another healthy donor (allogenic).

The first step in the production of CAR-T cells involves usingapheresis, e.g., leukocyte apheresis, to remove blood from a patient'sbody and separate the leukocytes. After a sufficient quantity ofleukocytes have been harvested, the leukapheresis product is enrichedfor T-cells, which involves washing the cells out of the leukapheresisbuffer. T-cell subsets having particular bio-markers are then isolatedfrom the enriched sub-population using specific antibody conjugates ormarkers.

After isolation of targeted T-cells, the cells are activated in acertain environment in which they can actively proliferate. For example,the cells may be activated using magnetic beads coated withanti-CD3/anti-CD28 monoclonal antibodies or cell-based artificialantigen presenting cells (aAPCs), which can be removed from the cultureusing magnetic separation. The T-cells are then transduced with CARgenes by either an integrating gammaretrovirus (RV) or by lentivirus(LV) vectors. The viral vector uses viral machinery to attach to thepatient cells, and, upon entry into the cells, the vector introducesgenetic material in the form of RNA. In the case of CAR-T cell therapy,this genetic material encodes the CAR. The RNA is reverse-transcribedinto DNA and permanently integrates into the genome of the patientcells; allowing CAR expression to be maintained as the cells divide andare grown to large numbers in a bioreactor. The CAR is then transcribedand translated by the patient cells, and the CAR is expressed on thecell surface.

After the T cells are activated and transduced with the CAR-encodingviral vector, the cells are expanded to large numbers in a bioreactor toachieve a desired cell density. After expansion, the cells areharvested, washed, concentrated and formulated for infusion into apatient.

Existing systems and methods for manufacturing an infusible dose of CART cells require many complex operations involving a large number ofhuman touchpoints, which adds time to the overall manufacturing processand increases the risk of contamination. While recent efforts toautomate the manufacturing process have eliminated some humantouchpoints, these systems still suffer from high cost, inflexibilityand workflow bottlenecks. In particular, systems utilizing increasedautomation are very costly and inflexible, in that they requirecustomers to adapt their processes to the particular equipment of thesystem.

In view of the above, there is a need for a bioprocessing system forcellular immunotherapies that reduces contamination risk by increasingautomation and decreasing human handling. In addition, there is a needfor a bioprocessing system for cell therapy manufacturing that balancesthe needs of flexibility in development and consistency in volumeproduction, as well as meets the desire for different customers to rundifferent processes.

BRIEF DESCRIPTION

Certain embodiments commensurate in scope with the originally claimedsubject matter are summarized below. These embodiments are not intendedto limit the scope of the claimed subject matter, but rather theseembodiments are intended only to provide a brief summary of the possibleembodiments. Indeed, the disclosure may encompass a variety of formsthat may be similar to or different from the embodiments set forthbelow.

In one embodiment, a bioprocessing system includes a first moduleconfigured for enriching and isolating a population of cells, a secondmodule configured for activating, genetically transducing and expandingthe population of cells, and a third module configured for harvestingthe expanded population of cells.

In another embodiment, a bioprocessing system includes a first moduleconfigured for enriching and isolating cells, a plurality of secondmodules, each second module configured for activating, geneticallytransducing and expanding the cells, and a third module configured forharvesting the cells after expansion. Each second module is configuredto support the activation, genetic transduction and expansion ofdifferent populations of cells in parallel with one another.

In another embodiment, a method of bioprocessing includes the steps ofin a first module, enriching and isolating a population of cells, in asecond module, activating, genetically transducing, and expanding thepopulation of cells, and in a third module, harvesting the expandedpopulation of cells. The steps of activating, genetically transducingand expanding the population of cells are carried out without removingthe population of cells from the second module.

In another embodiment, an apparatus for bioprocessing includes a housingand a drawer receivable within the housing. The drawer including aplurality of sidewalls and a bottom defining a processing chamber, and agenerally open top. The drawer is movable between a closed position inwhich the drawer is received within the housing, and an open position inwhich the drawer extends from the housing enabling access to theprocessing chamber through the open top. The apparatus also includes atleast one bed plate positioned within the processing chamber andconfigured to receive a bioreactor vessel.

In another embodiment, a method of bioprocessing includes the steps ofsliding a drawer having a plurality of sidewalls, a bottom and agenerally open top from a closed position within a housing to an openposition to extend the drawer from the housing, through the generallyopen top, positioning a bioreactor vessel, through the generally opentop, on a static bed plate positioned within the drawer, sliding thedrawer to the closed position, and controlling a drawer engagementactuator to engage a plurality of fluid flow lines with at least onepump and a plurality of pinch valve linear actuators.

In another embodiment, a system for bioprocessing includes a housing, afirst drawer receivable within the housing, the first drawer including aplurality of sidewalls and a bottom defining a first processing chamber,and a generally open top, at least one first bed plate positioned withinthe processing chamber of the first drawer and configured to receive orotherwise engage a first bioreactor vessel thereon, a second drawerreceivable within the housing in stacked relationship with the firstdrawer, the second drawer including a plurality of sidewalls and abottom defining a second processing chamber, and a generally open top,and at least one second bed plate positioned within the processingchamber of the second drawer and configured to receive or otherwiseengage a second bioreactor vessel thereon. The first drawer and thesecond drawer are each movable between a closed position in which thefirst drawer and/or the second drawer are received within the housing,and an open position in which the first drawer and/or the second drawerextends from the housing enabling access to the processing chambers,respectively, through the open top.

In yet another embodiment, an apparatus for bioprocessing includes ahousing, a drawer receivable within the housing, the drawer including aplurality of sidewalls and a bottom surface defining a processingchamber, and a generally open top, the drawer being movable between aclosed position in which the drawer is received within the housing, andan open position in which the drawer extends from the housing enablingaccess to the processing chamber through the open top, at least one bedplate positioned within the processing chamber adjacent to the bottomsurface, and a kit receivable within the processing chamber. The kitincludes a plurality of sidewalls and a bottom surface defining aninterior compartment, and a generally open top, an opening formed in thebottom surface of the kit, the opening having a perimeter, and abioreactor vessel positioned above the at least one opening within theinterior compartment and supported by the bottom surface such that aportion of the bioreactor vessel is accessible through the opening inthe bottom surface. The kit is receivable within the processing chambersuch that the bed plate extends through the opening in the bottomsurface of the tray to support the bioreactor vessel above the bottomsurface of the kit.

In yet another embodiment, a system for bioprocessing includes a trayhaving a plurality of sidewalls and a bottom surface defining aninterior compartment, and a generally open top, at least one openingformed in the bottom surface, the at least one opening having aperimeter, a first tubing holder block integrated with the tray andconfigured to receive at least one pump tube and hold the at least onepump tube in position for selective engagement with a pump, a secondtubing holder block integrated with the tray and configured to receive aplurality of pinch valve tubes and hold each pinch valve tube of theplurality of pinch valve tubes in position for selective engagement witha respective actuator of a pinch valve array, and a bioreactor vesselpositioned above the at least one opening within the interiorcompartment and supported by the bottom surface such that a portion ofthe bioreactor vessel is accessible through the opening in the bottomsurface.

In yet another embodiment, a system for bioprocessing includes aprocessing chamber having a plurality of sidewalls, a bottom surface,and a generally open top, a bed plate positioned within the processingchamber adjacent to the bottom surface, and a tray. The tray includes aplurality of sidewalls and a bottom surface defining an interiorcompartment, and a generally open top, and an opening in the bottomsurface of the tray, the opening having a perimeter. The perimeter ofthe opening is shaped and/or dimensioned such that a bioreactor vesselcan be positioned above the opening and supported by the bottom surfaceof the tray while a portion of the bioreactor vessel is accessiblethrough the opening in the bottom surface. The tray is receivable withinthe processing chamber such that the bed plate extends through theopening in the bottom surface of the tray to support the bioreactorvessel.

In yet another embodiment, a system for bioprocessing includes a trayhaving a plurality of sidewalls and a bottom surface defining aninterior compartment, and a generally open top, and at least one openingin the bottom surface, the opening bounded by a perimetrical edge,wherein the opening is shaped and/or dimensioned such that a bioreactorvessel can be positioned above the opening and supported by the bottomsurface of the tray within the interior compartment.

In yet another embodiment, a method of bioprocessing includes the stepsof placing a bioreactor vessel in a disposable tray, the disposable trayhaving a plurality of sidewalls and a bottom surface defining aninterior compartment, a generally open top, an opening formed in thebottom surface, and a plurality of tabs or projections extending intothe opening from the bottom surface, arranging the bioreactor vesselwithin the tray such that the bioreactor vessel is supported by theplurality of tabs above the opening, and placing the tray into aprocessing chamber having a bed plate such that the bed plate isreceived through the opening in the tray and supports the bioreactorvessel.

In yet another embodiment, a tubing module for a bioprocessing systemincludes a first tubing holder block configured to receive at least onepump tube and hold the at least one pump tube in position for selectiveengagement with a peristaltic pump, and a second tubing holder blockconfigured to receive a plurality of pinch valve tubes and hold eachpinch valve tube of the plurality of pinch valve tubes in position forselective engagement with a respective actuator of a pinch valve array.The first tubing holder block and the second tubing holder block areinterconnected.

In yet another embodiment, a system for bioprocessing includes a trayhaving a plurality of sidewalls and a bottom surface defining aninterior compartment, and a generally open top, the tray beingconfigured to receive, support or otherwise engage thereon a bioreactorvessel, a pump assembly positioned adjacent to the rear sidewall of thetray, a pinch valve array positioned adjacent to the rear sidewall ofthe tray, and a tubing module positioned at a rear of the tray. Thetubing module includes a first tubing holder block configured to receiveat least one pump tube and hold the at least one pump tube in positionfor selective engagement with the pump assembly, and a second tubingholder block configured to receive a plurality of pinch valve tubes andhold each pinch valve tube of the plurality of pinch valve tubes inposition for selective engagement with a respective actuator of thepinch valve array.

In yet another embodiment, a bioreactor vessel includes a bottom plate,a vessel body coupled to the bottom plate, the vessel body and thebottom plate defining an interior compartment therebetween, and aplurality of recesses formed in the bottom plate, each recess of theplurality of recesses being configured to receive a correspondingalignment pin on a bed plate for aligning the bioreactor vessel on thebed plate.

In yet another embodiment, a method for bioprocessing includesoperatively connecting a bottom plate to a vessel body to define aninterior compartment therebetween, the bottom plate and the vessel bodyforming a bioreactor vessel, aligning a recess in the bottom plate withan alignment pin of a bioprocessing system, and seating the bioreactorvessel on a bed plate of the bioprocessing system.

In yet another embodiment, a bioprocessing system includes a first fluidassembly having a first fluid assembly line connected to a first port ofa first bioreactor vessel though a first bioreactor line of a firstbioreactor vessel, the first bioreactor line of the first bioreactorvessel including a first bioreactor line valve for providing selectivefluid communication between the first fluid assembly and the first portof the first bioreactor vessel, a second fluid assembly having a secondfluid assembly line connected to a second port of the first bioreactorvessel through a second bioreactor line of the first bioreactor vessel,the second bioreactor line of the first bioreactor vessel including asecond bioreactor line valve for providing selective fluid communicationbetween the second fluid assembly and the second port of the firstbioreactor vessel, and an interconnect line providing for fluidcommunication between the first fluid assembly and the second fluidassembly, and for fluid communication between the second bioreactor lineof the first bioreactor vessel and the first bioreactor line of thefirst bioreactor vessel.

In yet another embodiment a method of bioprocessing includes providing afirst fluid assembly having a first fluid assembly line connected to afirst port of a first bioreactor vessel through a first bioreactor lineof the first bioreactor vessel, providing a second fluid assembly havinga second fluid assembly line connected to a second port of the firstbioreactor vessel through a second bioreactor line of the firstbioreactor vessel, and providing an interconnect line between the secondbioreactor line of the first bioreactor vessel and the first bioreactorline of the first bioreactor vessel, the interconnecting line allowingfor fluid communication between the first fluid assembly and the secondfluid assembly, and for fluid communication between the secondbioreactor line of the first bioreactor vessel and the first bioreactorline of the first bioreactor vessel.

In yet another embodiment, a bioprocessing method for cell therapyincludes genetically modifying a population of cells in a bioreactorvessel to produce a population of genetically modified cells, andexpanding the population of genetically modified cells within thebioreactor vessel to generate a number of genetically modified cellssufficient for one or more doses for use in a cell therapy treatmentwithout removing the population of genetically modified cells from thebioreactor vessel.

In yet another embodiment, a bioprocessing method includes coating abioreactor vessel with a reagent for enhancing efficiency of geneticmodification of a population of cells, genetically modifying cells of apopulation of cells to produce a population of genetically modifiedcells, and expanding the population of genetically modified cells in thebioreactor vessel without removing the genetically modified cells fromthe bioreactor vessel.

In yet another embodiment, a bioprocessing method includes activatingcells of a population of cells in a bioreactor vessel using magnetic ornon-magnetic beads to produce a population of activated cells,genetically modifying the activated cells in the bioreactor vessel toproduce a population of genetically modified cells, washing thegenetically modified cells in the bioreactor vessel to remove unwantedmaterials, and expanding the population of genetically modified cells inthe bioreactor vessel to produce an expanded population of transducedcells. Activating, genetically modifying, washing, and expanding arecarried out in the bioreactor vessel without removing the cells from thebioreactor vessel.

In yet another embodiment, a kit for use in a bioprocessing systemincludes a process bag, a source bag, a bead addition vessel and aprocess loop configured to be in fluid communication with the processbag, the source bag and the bead addition vessel. The process loopadditionally includes pump tubing configured to in fluid communicationwith a pump.

In yet another embodiment, an apparatus for bioprocessing includes a kitcomprising a process bag, a source bag, and a bead addition vesselconfigured to be in fluid communication with a process loop, the processloop additionally comprising pump tubing configured to in fluidcommunication with a pump, a magnetic field generator configured togenerate a magnetic field, a plurality of hooks for suspending thesource bag, the process bag, and the bead addition vessel, each hook ofthe plurality of hooks is operatively connected to a load cellconfigured to sense a weight of the bag connected thereto, at least oneair bubble sensor, and a pump configured to be in fluid communicationwith the process loop.

In an embodiment, a method of bioprocessing includes combining asuspension comprising a population of cells with magnetic beads to forma population of bead-bound cells in the suspension, isolating thepopulation of bead-bound cells on a magnetic isolation column, andcollecting target cells from the population of cells.

In an embodiment, a non-transitory computer readable medium is provided.The non-transitory computer readable medium includes instructionsconfigured to adapt a controller to maintain a first target environmentin a bioreactor vessel containing a population of cells for a firstincubation period to produce a population of genetically modified cellsfrom the population of cells, initiate a flow of media to the bioreactorvessel, maintain a second target environment in the bioreactor vesselfor a second incubation period to produce an expanded population ofgenetically modified cells.

In another embodiment, a non-transitory computer readable medium isprovided. The non-transitory computer readable medium includesinstructions configured to adapt a controller to maintain a first targetenvironment in a first bioreactor vessel for a first incubation periodto activate a population of cells in the first bioreactor, and maintaina second target environment in the first bioreactor vessel for a secondincubation period to produce a population of genetically modified cellsfrom the population of cells.

In yet another embodiment, a non-transitory computer readable medium isprovided. The non-transitory computer readable medium includesinstructions configured to adapt a controller to receive data relatingto a mass and/or volume of a bioreactor vessel containing a populationof cells suspended in a culture medium, actuate a first pump to pumpfresh media to the bioreactor vessel, actuate a second pump to pumpspent media from the bioreactor vessel to a waste bag, and control anoperational setpoint of at least one of the first pump and the secondpump in dependence upon the data relating to the mass and/or volume ofthe bioreactor vessel.

DRAWINGS

The present invention will be better understood from reading thefollowing description of non-limiting embodiments, with reference to theattached drawings, wherein below:

FIG. 1 is a schematic illustration of a bioprocessing system accordingto an embodiment of the invention.

FIG. 2 is a schematic illustration of a bioprocessing system accordingto another embodiment of the invention.

FIG. 3 is a block diagram illustrating the fluid flowconfiguration/system of a cell activation, genetic modification andexpansion subsystem of the bioprocessing system of FIG. 1.

FIG. 4 is a detail view of a portion of the block diagram of FIG. 3,illustrating a first fluid assembly of the fluid flowconfiguration/system.

FIG. 5 is a detail view of a portion of the block diagram of FIG. 3,illustrating a second fluid assembly of the fluid flowconfiguration/system.

FIG. 6 is a detail view of a portion of the block diagram of FIG. 3,illustrating a sampling assembly of the fluid flow configuration/system.

FIG. 7 is a detail view of a portion of the block diagram of FIG. 3illustrating a filtration flowpath of the fluid flowconfiguration/system.

FIG. 8 is a perspective view of a bioreactor vessel according to anembodiment of the invention.

FIG. 9 is an exploded view of the bioreactor vessel of FIG. 8.

FIG. 10 is an exploded, cross-sectional view of the bioreactor vessel ofFIG. 8.

FIG. 11 is an exploded bottom, perspective view of the bioreactor vesselof FIG. 8.

FIG. 12 is a perspective top and front view of a disposable drop-in kitof the bioprocessing system of FIG. 1, according to an embodiment of theinvention.

FIG. 13 is another perspective top and front view of the disposabledrop-in kit of FIG. 12.

FIG. 14 is a perspective top and rear view of the disposable drop-in kitof FIG. 12.

FIG. 15 is a perspective view of a tray of the disposable drop-in kit ofFIG. 12, according to an embodiment of the invention.

FIG. 16 is a front perspective view of a tubing module of the disposabledrop-in kit of FIG. 12, according to an embodiment of the invention.

FIG. 17 is a rear perspective view of the tubing module of FIG. 16.

FIG. 18 is an elevational view of a second tubing holder block of thetubing module, according to an embodiment of the invention.

FIG. 19 is a cross-sectional view of the second tubing holder block ofFIG. 18,

FIG. 20 is another perspective front view of the drop-in kit of FIG. 12,showing the flow architecture integrated therein.

FIG. 21 is a perspective rear view of the drop-in kit of FIG. 12,showing the flow architecture integrated therein.

FIG. 22 is a front elevational view of the drop-in kit of FIG. 12,showing the flow architecture integrated therein.

FIG. 23 is a perspective view of a bioprocessing apparatus, according toan embodiment of the invention.

FIG. 24 is a perspective view of a drawer of the bioprocessing apparatusfor receiving the drop-in kit of FIG. 12, according to an embodiment ofthe invention.

FIG. 25 is a top plan view of the drawer of FIG. 24.

FIG. 26 is a front, perspective view of a processing chamber of thedrawer of FIG. 24.

FIG. 27 is a top plan view of the processing chamber of the drawer.

FIG. 28 is a top plan view of a bed plate of the bioprocessing apparatusof FIG. 23.

FIG. 28A is a top plan view of the hardware components housed beneaththe bed plate of FIG. 28.

FIG. 29 is a side elevational view of the bioprocessing apparatus ofFIG. 12.

FIG. 30 is a perspective view of a drawer engagement actuator of thebioprocessing apparatus of FIG. 12.

FIG. 31 is a top plan view of the drawer of the bioprocessing apparatus,illustrating a clearance position of a drawer engagement actuator, pumpassembly and solenoid array.

FIG. 32 is a top plan view of the drawer of the bioprocessing apparatus,illustrating an engagement position of the drawer engagement actuator,pump assembly and solenoid array.

FIG. 33 is a perspective view of the bioprocessing apparatus,illustrating the drop-in kit in position within the processing chamberof the drawer.

FIG. 34 is a top plan view of the bioprocessing apparatus, illustratingthe drop-in kit in position within the processing chamber of the drawer.

FIG. 35 is a perspective view of a peristaltic pump assembly of thebioprocessing apparatus.

FIG. 36 is a side elevational view of the peristaltic pump assembly anda tubing holder module of the drop-in kit, illustrating the relationshipbetween components.

FIG. 37 is a perspective view of a solenoid array and pinch valve anvilswhich form a pinch valve array of the bioprocessing apparatus.

FIG. 38 is another perspective view of the pinch valve array of thebioprocessing apparatus.

FIG. 39 is another perspective view of the pinch valve array,illustrating positioning of the tubing holder module of the drop-in kitwith respect to the pinch valve array, in an engaged position.

FIG. 40 is a cross-sectional view of the drawer of the bioprocessingapparatus, illustrating a seated position of the bioreactor vessel onthe bed plate.

FIG. 41 is a side elevational view of a bioreactor received on a bedplate, illustrating an agitation/mixing mode of operation of thebioreactor system.

FIG. 42 is a side, cross sectional view of the bioreactor received onthe bed plate, illustrating the agitation/mixing mode of operation ofthe bioreactor system.

FIG. 43 is a schematic illustration of the bioreactor vessel shown afluid level within the bioreactor vessel during the agitation/mixingmode of operation.

FIG. 44 is a cross-sectional, detail view of an interface betweenlocating pins on the bed plate and receiving recesses on a bioreactorvessel during agitation/mixing mode of operation.

FIG. 45 is a perspective view of a bioprocessing apparatus having aflip-down front panel according to an embodiment of the invention,showing the processing drawer thereof in an open position.

FIG. 46 is another perspective view of the bioprocessing apparatus ofFIG. 45, showing the processing drawer thereof in an open position.

FIG. 47 is an enlarged, perspective view of an auxiliary compartment ofthe bioprocessing apparatus of FIG. 45, showing the processing drawer ina closed position with access to the auxiliary compartment.

FIG. 48 is another enlarged, perspective view of the auxiliarycompartment of the bioprocessing apparatus of FIG. 45, showing theprocessing drawer in the closed position with access to the auxiliarycompartment.

FIG. 49 is a perspective view of the bioprocessing apparatus of FIG. 45,showing the processing drawer thereof in the closed position with accessto the auxiliary compartment.

FIG. 50 is another perspective view of the bioprocessing apparatus ofFIG. 45, showing the processing drawer thereof in the closed positionwith access to the auxiliary compartment.

FIG. 51 is a perspective view of the auxiliary compartment of thebioprocessing apparatus, according to another embodiment of theinvention.

FIG. 52 is a perspective view of a bioprocessing system having a wastetray, according to an embodiment of the invention.

FIGS. 53-77 are schematic illustrations of an automated, genericprotocol of the bioprocessing system utilizing the fluid flowarchitecture of FIG. 3, according to an embodiment of the invention.

FIG. 78 is perspective view of an enrichment and isolation apparatusaccording to an embodiment of the invention.

FIG. 79 is a process flow diagram of the enrichment and isolationapparatus of FIG. 78.

FIG. 80 is a schematic illustration of the fluid flow architecture ofthe apparatus of FIG. 78, for carrying out enrichment and isolation of apopulation of cells.

FIG. 81 is a flowchart of a method of bioprocessing using the system ofFIG. 1, in accordance with an embodiment of the present invention.

DETAILED DESCRIPTION

Reference will be made below in detail to exemplary embodiments of theinvention, examples of which are illustrated in the accompanyingdrawings. Wherever possible, the same reference characters usedthroughout the drawings refer to the same or like parts.

As used herein, the term “flexible” or “collapsible” refers to astructure or material that is pliable, or capable of being bent withoutbreaking, and may also refer to a material that is compressible orexpandable. An example of a flexible structure is a bag formed ofpolyethylene film. The terms “rigid” and “semi-rigid” are used hereininterchangeably to describe structures that are “non-collapsible,” thatis to say structures that do not fold, collapse, or otherwise deformunder normal forces to substantially reduce their elongate dimension.Depending on the context, “semi-rigid” can also denote a structure thatis more flexible than a “rigid” element, e.g., a bendable tube orconduit, but still one that does not collapse longitudinally undernormal conditions and forces.

A “vessel,” as the term is used herein, means a flexible bag, a flexiblecontainer, a semi-rigid container, a rigid container, or a flexible orsemi-rigid tubing, as the case may be. The term “vessel” as used hereinis intended to encompass bioreactor vessels having a wall or a portionof a wall that is semi-rigid or rigid, as well as other containers orconduits commonly used in biological or biochemical processing,including, for example, cell culture/purification systems, mixingsystems, media/buffer preparation systems, and filtration/purificationsystems, e.g., chromatography and tangential flow filter systems, andtheir associated flow paths. As used herein, the term “bag” means aflexible or semi-rigid container or vessel used, for example, ascontainment device for various fluids and/or media.

As used herein, “fluidly coupled” or “fluid communication” means thatthe components of the system are capable of receiving or transferringfluid between the components. The term fluid includes gases, liquids, orcombinations thereof. As used herein, “electrical communication” or“electrically coupled” means that certain components are configured tocommunicate with one another through direct or indirect signaling by wayof direct or indirect electrical connections. As used herein,“operatively coupled” refers to a connection, which may be direct orindirect. The connection is not necessarily a mechanical attachment.

As used herein, the term “tray” refers to any object, capable of atleast temporarily supporting a plurality of components. The tray may bemade of a variety of suitable materials. For example, the tray may bemade of cost-effective materials suitable for sterilization andsingle-use disposable products.

As used herein, the term “functionally-closed system” refers to aplurality of components that make up a closed fluid path that may haveinlet and outlet ports, to add or remove fluid or air from the system,without compromising the integrity of the closed fluid path (e.g. tomaintain an internally sterile biomedical fluid path), whereby the portsmay comprise, for example, filters or membranes at each port to maintainthe sterile integrity when fluids or air is added or removed from thesystem. The components, depending on a given embodiment, may comprisebut are not limited to, one or more conduits, valves (e.g. multiportdiverters), vessels, receptacles, and ports.

Embodiments of the invention provide systems and methods formanufacturing cellular immunotherapies from a biological sample (e.g.,blood, tissue, etc.). In some embodiments, an apparatus for fluid linemanagement in a bioprocessing system is provided. An apparatus for fluidline management may include a first tubing holder block configured toreceive at least one pump tube and hold the at least one pump tube inposition for selective engagement with a peristaltic pump, and a secondtubing holder block configured to receive a plurality of pinch valvetubes and hold each pinch valve tube of the plurality of pinch valvetubes in position for selective engagement with a respective actuator ofa pinch valve array. In embodiments, the apparatus may be configured foruse with a bioprocessing system having a tray having a plurality ofsidewalls and a bottom surface defining an interior compartment, and agenerally open top, a pump assembly positioned adjacent to the rearsidewall of the tray, and a pinch valve array positioned adjacent to therear sidewall of the tray such that the fluid line management apparatusholds the at least one pump tube in position for selective engagementwith the pump assembly, and holds the pinch valve tubes in position forselective engagement with actuators of the pinch valve array.

With reference to FIG. 1, a schematic illustration of a bioprocessingsystem 10 according to an embodiment of the invention is illustrated.The bioprocessing system 10 is configured for use in the manufacture ofcellular immunotherapies (e.g., autologous cellular immunotherapies),where, for example, human blood, fluid, tissue, or cell sample iscollected, and a cellular therapy is generated from or based on thecollected sample. One type of cellular immunotherapy that can bemanufactured using the bioprocessing system 10 is chimeric antigenreceptor (CAR) T cell therapy, although other cellular therapies mayalso be produced using the system of the invention or aspects thereofwithout departing from the broader aspects of the invention. Asillustrated in FIG. 1, the manufacture of a CAR T cell therapy generallybegins with collection of a patient's blood and separation of thelymphocytes through apheresis. Collection/apheresis may take place in aclinical setting, and the apheresis product is then sent to a laboratoryor manufacturing facility for production of CAR T-cells. In particular,once the apheresis product is received for processing, a desired cellpopulation (e.g., white blood cells) is enriched for or separated fromthe collected blood for manufacturing the cellular therapy, and targetcells of interest are isolated from the initial cell mixture. The targetcells of interest are then activated, genetically modified tospecifically target and destroy tumor cells, and expanded to achieve adesired cell density. After expansion, the cells are harvested, and adose is formulated. The formulation is often then cryopreserved anddelivered to a clinical setting for thawing, preparation and, finally,infusion into the patient.

With further reference to FIG. 1, the bioprocessing system 10 of theinvention includes a plurality of distinct modules or subsystems thatare each configured to carry out a particular subset of manufacturingsteps in a substantially automated, functionally-closed and scalablemanner. In particular, the bioprocessing system 10 includes a firstmodule 100 configured to carry out the steps of enrichment andisolation, a second module 200 configured to carry out the steps ofactivation, genetic modification and expansion, and a third module 300configured to carry out the step of harvesting the expanded cellpopulation. In an embodiment, each module 100, 200, 300 may becommunicatively coupled to a dedicated controller (e.g., firstcontroller 110, second controller 210, and third controller 310,respectively). The controllers 110, 210 and 310 are configured toprovide substantially automated control over the manufacturing processeswithin each module. While the first module 100, second module 200 andthird module 300 are illustrated as including dedicated controllers forcontrolling the operation of each module, it is contemplated that amaster control unit may be utilized to provide global control over thethree modules. Each module 100, 200, 300 is designed to work in concertwith the other modules to form a single, coherent bioprocessing system10, as discussed in detail below.

By automating the processes within each module, product consistency fromeach module can be increased and costs associated with extensive manualmanipulations reduced. In addition, as discussed in detail hereinafter,each module 100, 200, 300 is substantially closed, which helps ensurepatient safety by decreasing the risk of outside contamination, ensuresregulatory compliance, and helps avoid the costs associated with opensystems. Moreover, each module 100, 200, 300 is scalable, to supportboth development at low patient numbers and commercial manufacturing athigh patient numbers.

With further reference to FIG. 1, the particular manner in which theprocess steps are compartmentalized in distinct modules that eachprovide for closed and automated bioprocessing allows for efficientutilization of capital equipment to an extent heretofore not seen in theart. As will be appreciated, the step of expanding the cell populationto achieve a desired cell density prior to harvest and formulation istypically the most time-consuming step in the manufacturing process,while the enrichment and isolation steps, and the harvesting andformulation steps, as well as activation and genetic modification steps,are much less time consuming. Accordingly, attempts to automate theentire cell therapy manufacturing process, in addition to beinglogistically challenging, can exacerbate bottlenecks in the process thathamper workflow and decrease manufacturing efficiency. In particular, ina fully-automated process, while the steps of enrichment, isolation,activation and genetic modification of cells can take place ratherquickly, expansion of the genetically modified cells takes place veryslowly. Accordingly, manufacture of a cellular therapy from a firstsample (e.g., the blood of a first patient) would progress quickly untilthe expansion step, which requires a substantial amount of time toachieve a desired cell density for harvest. With a fully automatedsystem, the entire process/system would be monopolized by the expansionequipment performing expansion of the cells from the first sample, andprocessing of a second sample could not begin until the entire systemwas freed up for use. In this respect, with a fully-automatedbioprocessing system, the entire system is essentially offline andunavailable for processing of a second sample until the entire celltherapy manufacturing process, from enrichment to harvest/formulation iscompleted on the first sample.

Embodiments of the invention, however, allow for parallel processing ofmore than one sample (from the same or different patients) to providefor more efficient utilization of capital resources. This advantage is adirect result of the particular manner in which the process steps areseparated into the three modules 100, 200, 300, as alluded to above.With particular reference to FIG. 2, in an embodiment, a single firstmodule 100 and/or a single third module 300 can be utilized inconjunction with multiple second modules, e.g., second modules 200 a,200 b, 200 c, in a bioprocessing system 12, to provide for parallel andasynchronous processing of multiple samples from the same or differentpatients. For example, a first apheresis product from a first patientmay be enriched and isolated using the first module 100 to produce afirst population of isolated target cells, and the first population oftarget cells may then be transferred to one of the second modules, e.g.,module 200 a, for activation, genetic modification and expansion undercontrol of controller 210 a. Once the first population of target cellsis transferred out of the first module 100, the first module is againavailable for use to process a second apheresis product from, forexample, a second patient. A second population of target cells producedin the first module 100 from the sample taken from the second patientcan then be transferred to another second module, e.g., second module200 b, for activation, genetic modification and expansion under controlof controller 201 b.

Similarly, after the second population of target cells is transferredout of the first module 100, the first module is again available for useto process a third apheresis product from, for example, a third patient.A third target population of cells produced in the first module 100 fromthe sample taken from the third patient can then be transferred toanother second module, e.g., second module 200 c, for activation,genetic modification and expansion under control of controller 201 c. Inthis respect, expansion of, for example, CAR-T cells for a first patientcan occur simultaneously with the expansion of CAR-T cells for a secondpatient, a third patient, etc.

This approach also allows the post processing to occur asynchronously asneeded. In other words, patient cells may not all grow at the same time.The cultures may reach the final density at different times, but themultiple second modules 200 are not linked, and the third module 300 canbe used as needed. With the present invention, while samples can beprocessed in parallel, they do not have to be done in batches.

Harvesting of the expanded populations of cells from the second modules200 a, 200 b and 200 c can likewise be accomplished using a single thirdmodule 300 when each expanded populations of cells are ready forharvest.

Accordingly, by separating the steps of activation, genetic modificationand expansion, which is the most time consuming, and which share certainoperational requirements and/or require similar culture conditions, intoa stand-alone, automated and functionally-closed module, the othersystem equipment that is utilized for enrichment, isolation, harvest andformulation is not tied up or offline while expansion of one populationof cells is carried out. As a result, the manufacture of multiple celltherapies may be carried out simultaneously, maximizing equipment andfloorspace usage and increasing overall process and facility efficiency.It is envisioned that additional second modules may be added to thebioprocessing system 10 to provide for the parallel processing of anynumber of cell populations, as desired. Accordingly, the bioprocessingsystem of the invention allows for plug-and-play like functionality,which enables a manufacturing facility to scale up or scale down withease.

In an embodiment, the first module 100 may be any system or devicecapable of producing, from an apheresis product taken from a patient, atarget population of enriched and isolated cells for use in a biologicalprocess, such as the manufacture of immunotherapies and regenerativemedicines. For example, the first module 100 may be a modified versionof a Sefia Cell Processing System, available from GE Healthcare. Theconfiguration of the first module 100 according to some embodiments ofthe invention is discussed in detail hereinafter.

In an embodiment, the third module 300 may similarly be any system ordevice capable of harvesting and/or formulating CAR-T cells or othermodified cells produced by the second module 200 for infusion into apatient, for use in cellular immunotherapies or regenerative medicine.In some embodiments, the third module 300 may likewise be a Sefia CellProcessing System, available from GE Healthcare. In some embodiments,the first module 100 may first be utilized for enrichment and isolationof cells (which are then transferred to the second module 200 foractivation, transduction and expansion (and in some embodiments,harvesting)), and then also used at the end of the process for cellharvesting and/or formulation. In this respect, in some embodiments, thesame equipment can be utilized for the front-end cell enrichment andisolation steps, as well as the back-end harvesting and/or formulationsteps.

Focusing first on the second module 200, the ability to combine theprocess steps of cell activation, genetic modification and cellexpansion in a single, functionally-closed and automated module 200 thatprovides for the workflow efficiencies described above is enabled by thespecific configuration of components within the second module 200, and aunique flow architecture that provides for a specific interconnectivitybetween such components. FIGS. 3-77, discussed below, illustrate variousaspects of the second module 200 according to various embodiments of theinvention. Referring first to FIG. 3, a schematic illustrating the fluidflow architecture 400 (also broadly referred to herein as bioprocessingsubsystem 400 or bioprocessing system 400) within the second module 200that provides for cell activation, genetic modification and expansion(an in some cases, harvesting), is shown. The system 400 includes afirst bioreactor vessel 410 and a second bioreactor vessel 420. Thefirst bioreactor vessel includes at least a first port 412 and a firstbioreactor line 414 in fluid communication with the first port 412, anda second port 416 and a second bioreactor line 418 in fluidcommunication with the second port 416. Similarly, the second bioreactorvessel includes at least a first port 422 and a first bioreactor line424 in fluid communication with the first port 422, and a second port426 and a second bioreactor line 428 in fluid communication with thesecond port 426. Together, the first bioreactor vessel 410 and secondbioreactor vessel 420 form a bioreactor array 430. While the system 400is shown as having two bioreactor vessels, embodiments of the inventionmay include a single bioreactor or more than two bioreactor vessels.

The first and second bioreactor lines 414, 418, 424, 428 of the firstand second bioreactor vessels 410, 420 each include a respective valvefor controlling a flow of fluid therethrough, as discussed hereinafter.In particular, the first bioreactor line 414 of the first bioreactorvessel 410 includes a first bioreactor line valve 432, while the secondbioreactor line 418 of the first bioreactor vessel 410 includes a secondbioreactor line valve 424. Similarly, the first bioreactor line 424 ofthe second bioreactor vessel 420 includes a first bioreactor line valve436, while the second bioreactor line 428 of the second bioreactorvessel 420 includes a second bioreactor line valve 438.

With further reference to FIG. 3, the system 400 also includes a firstfluid assembly 440 having a first fluid assembly line 442, a secondfluid assembly 444 having a second fluid assembly line 446, and asampling assembly 448. An interconnect line 450 having an interconnectline valve 452 provides for fluid communication between the first fluidassembly 440 and the second fluid assembly 444. As shown in FIG. 3, theinterconnect line 450 also provides for fluid communication between thesecond bioreactor line 418 and first bioreactor line 414 of the firstbioreactor vessel 410, allowing for circulation of a fluid along a firstcirculation loop of the first bioreactor vessel. Similarly, theinterconnect line also provides for fluid communication between thesecond bioreactor line 428 and first bioreactor line 424 of the secondbioreactor vessel 420, allowing for circulation of a fluid along asecond circulation loop of the second bioreactor vessel. Moreover, theinterconnect line 450 further provides for fluid communication betweenthe second port 416 and second bioreactor line 418 of the firstbioreactor vessel 410, and the first port 422 and first bioreactor line424 of the second bioreactor vessel 420, allowing for the transfer ofcontents of the first bioreactor vessel 410 to the second bioreactorvessel 420, as discussed hereinafter. As illustrated in FIG. 3, theinterconnect line 450, in an embodiment, extends from the secondbioreactor lines 418, 428 to the intersection of the first bioreactorline 414 of the first bioreactor vessel 410 and the first fluid assemblyline 442.

As illustrated by FIG. 3, the first and second fluid assemblies 440, 450are disposed along the interconnect line 450. Additionally, in anembodiment, the first fluid assembly is in fluid communication with thefirst port 412 of the first bioreactor vessel 410 and the first port ofthe second bioreactor vessel 420 through the first bioreactor line 414of the first bioreactor vessel and the first bioreactor line 424 of thesecond bioreactor vessel 420, respectively. The second fluid assembly444 is in fluid communication with the second port 416 of the firstbioreactor vessel 410 and the second port 426 of the second bioreactorvessel 420 via the interconnect line 450.

A first pump or interconnect line pump 454 capable of providing forbi-directional fluid flow is disposed along the first fluid assemblyline 442, and a second pump or circulation line pump 456 capable ofproviding for bi-directional fluid flow is disposed along theinterconnect line 450, the function and purpose of which will bediscussed below. In an embodiment, the pumps 454, 456 are high dynamicrange pumps. As also shown in FIG. 3, a sterile air source 458 isconnected to the interconnect line 450 through a sterile air source line460. A valve 462 positioned along the sterile air source line 460provides for selective fluid communication between the sterile airsource 458 and the interconnect line 450. While FIG. 3 shows the sterileair source 458 connected to the interconnect line 450, in otherembodiments the sterile air source may be connected to the first fluidassembly 440, the second fluid assembly 444, or the fluid flowpathintermediate the second bioreactor line valve and the first bioreactorline valve of either the first bioreactor or the second bioreactor,without departing from the broader aspects of the invention.

With additional reference now to FIGS. 4-6, detailed views of the firstfluid assembly 440, second fluid assembly 444 and sampling assembly 448are shown. With specific reference to FIG. 4, the first fluid assembly440 includes a plurality of tubing tails 464 a-f, each of which isconfigured for selective/removable connection to one of a plurality offirst reservoirs 466 a-f. Each tubing tail 464 a-f of the first fluidassembly 440 includes a tubing tail valve 468 a-f for selectivelycontrolling a flow of fluid to or from a respective one of the pluralityof first reservoirs 466 a-f of the first fluid assembly 440. While FIG.4 specifically shows that the first fluid assembly 440 includes sixfluid reservoirs, more or fewer reservoirs may be utilized to providefor the input or collection of various processing fluids, as desired. Itis contemplated that each tubing tail 464 a-f may be individuallyconnected to a reservoir 466 a-f, respectively, at a time requiredduring operation of fluid assembly 440, as described below.

With specific reference to FIG. 5, the second fluid assembly 444includes a plurality of tubing tails 470 a-d, each of which isconfigured for selective/removable connection to one of a plurality ofsecond reservoirs 472 a-d Each tubing tail 470 a-d of the second fluidassembly 444 includes a tubing tail valve 474 a-e for selectivelycontrolling a flow of fluid to or from a respective one of the pluralityof second reservoirs 472 a-d of the first fluid assembly 444. While FIG.5 specifically shows that the second fluid assembly 444 includes fourfluid reservoirs, more or fewer reservoirs may be utilized to providefor the input or collection of various processing fluids, as desired. Inan embodiment, at least one of the second reservoirs, e.g., secondreservoir 472 d is a collection reservoir for collecting an expandedpopulation of cells, as discussed hereinafter. In an embodiment, thesecond reservoir 472 a is a waste reservoir, the purpose of which isdiscussed below. The invention further contemplates that one or morereservoirs 472 a-d may be pre-connected to their respective tails 470a-d, with each additional reservoir being connected to its respectivetail in time for its use within the second fluid assembly 440.

In an embodiment, the first reservoirs 466 a-f and the second reservoirs472 a-d are single use/disposable, flexible bags. In an embodiment, thebags are substantially two-dimensional bags having opposing panelswelded or secured together about their perimeters and supportingconnecting conduit for connection to its respective tail, as is known inthe art.

In an embodiment, the reservoirs/bags may be connected to the tubingtails of the first and second tubing assembly using a sterile weldingdevice. In an embodiment, a welding device can be positioned next to themodule 200, and the welding device utilized to splice-weld one of thetubing tails to tail to the tube on the bag (while maintainingsterility). Thus the operator can provide the bag at the time it isneeded (e.g., by grabbing a tubing tail and inserting its free end intothe welding device, laying the bag tube's free end adjacent to the endof the tubing tail, cutting the tubes with a fresh razor blade, andheating the cut ends as the razor is pulled away while the two tube endsare forced together while still melted so that they re-solidifytogether). Conversely, a bag can be removed by welding the line from thebag and cutting at the weld to separate the two closed lines.Accordingly, the reservoirs/bags may be individually connected whendesired, and the present invention does not require that allreservoirs/bags must be connected at the beginning of a protocol, as anoperator will have access to the appropriate tubing tails during theentire process to connect a reservoir/bag in time for its use. Indeed,while it is possible that all reservoirs/bags are pre-connected, theinvention does not require pre-connection, and one advantage of thesecond module 200 is that it allows the operator to access the fluidassemblies/lines during operations so that spent bags may be connectedin a sterile manner, and disconnected so that other bags can besterilely connected during a protocol, as discussed below.

As illustrated in FIG. 6, the sampling assembly 448 includes one or moresampling lines, e.g., sampling lines 476 a-476 d, fluidly connected tothe interconnect line 450. Each of the sample lines 476 a-476 d mayinclude a sample line valve 478 a-d that is selectively actuatable toallow fluid to flow from the interconnect line 450 through the samplelines 476 a-476 d. As also shown therein, a distal end of each samplingline 476 a-476 d is configured for selective connection to a samplecollection device (e.g., sample collection devices 280 a and 280 d) forcollection of the fluid from the interconnect line 450. The samplecollection devices may take the form of any sampling device known in theart such as, for example, a syringe, dip tube, bag, etc. While FIG. 6illustrates the sampling assembly 448 being connected to theinterconnect line, in other embodiments the sampling assembly may befluidly coupled to the first fluid assembly 440, the second fluidassembly 444 a fluid flowpath intermediate the second bioreactor linevalve 434 and the first bioreactor line valves 432 of the firstbioreactor vessel 410, and/or a fluid flowpath intermediate the secondbioreactor line valve 438 and the first bioreactor line valve 436 of thesecond bioreactor vessel 420. The sampling assembly 448 provides forfully functionally-closed sampling of a fluid at one or more points inthe system 400, as desired.

Referring back to FIG. 3, in an embodiment, the system 400 may alsoinclude a filtration line 482 that is connected at two points along theinterconnect line 450 and defines a filtration loop along theinterconnect line 450. A filter 484 is positioned along the filtrationline 482 for removing permeate waste from a fluid passing through thefiltration line 482. As shown therein, the filtration line 482 includesan upstream filtration line valve 486 and a downstream filtration linevalve 488 positioned on the upstream and downstream side of the filter484, respectively. A waste line 490 provides fluid communication betweenthe filter 484 and the second fluid assembly 444 and, in particular,with tubing tail 470 a of the second fluid assembly 444, which isconnected to the waste reservoir 472 a. In this respect, the waste line490 conveys waste removed from the fluid passing through the filtrationline 482 by the filter 484 to the waste reservoir 472 a. As illustratedin FIG. 3, the filtration line 482 surrounds the interconnect line valve452 so that a flow of fluid through the interconnect line 450 can beforced through the filtration line 482, as discussed hereinafter. Apermeate pump 492 positioned along the waste line 490 is operable topump the waste removed by the filter to the waste reservoir 472 a. In anembodiment, the filter 484 is desirably an elongate hollow fiber filter,although other tangential-flow or cross-flow filtration means known inthe art such as, for example, a flat sheet membrane filter, may also beutilized without departing from the broader aspects of the invention.

In an embodiment, the valves of the first fluid assembly 440 and secondfluid assembly 444, as well as the bioreactor line valves (i.e., valves432, 434, 436, 438, sterile line valve 462, interconnect line valve 452and filtration line valves 486, 488 are pinch valves constructed in themanner hereinafter described. In an embodiment, the lines themselvesneed not include the pinch valves, and the depiction of the pinch valvesin FIGS. 3-8 may simply denote locates where pinch valves can operate onthe lines so as to prevent fluid flow. In particular, as discussed belowthe pinch valves of the flow architecture 400 may be provided byrespective actuators (e.g., solenoids) that operate/act against acorresponding anvil while the fluid path/line is in between to “pinchoff” the line to prevent fluid flow therethrough.

In an embodiment, the pumps 454, 456 and 492 are peristaltic pumps, andthe pumps are integrated into a single assembly, as discussedhereinafter. Desirably, operation of these valves and pumps areautomatically directed according to a programmed protocol so as toenable proper operation of module 200. It is contemplated that secondcontroller 210 may direct the operation of these valves and pumps bymodule 200.

Turning now to FIGS. 8-11, the configuration of the first bioreactorvessel 410 according to an embodiment of the invention is illustrated.As the second bioreactor vessel 420 is desirably, although not requiredto be, identical in configuration to the first bioreactor vessel 410,for simplicity, only first bioreactor vessel 410 will be describedbelow. In an embodiment, the bioreactor vessels 410, 420 areperfusion-enabled, silicone membrane-based bioreactor vessels thatsupport activation, transduction and expansion of a population of cellstherein. The bioreactor vessels 410, 420 may be used for cell culture,cell processing, and/or cell expansion to increase cell density for usein medical therapeutics or other processes. While the bioreactor vesselmay be disclosed herein as being used in conjunction with particularcell types, it should be understood that the bioreactor vessel may beused for activation, genetic modification and/or expansion of anysuitable cell type. Further, the disclosed techniques may be used inconjunction with adherent cells, i.e., cells that adhere to and/orproliferate on a cell expansion surface. In an embodiment, the first andsecond bioreactor vessels 410, 420 may be constructed and function asdisclosed in U.S. patent Ser. No. 15/893,336, filed on Feb. 9, 2018,which is incorporated by reference herein in its entirety.

As shown in FIGS. 8 and 9, the first bioreactor vessel 410 may include abottom plate 502 and a vessel body 504 coupled to the bottom plate 502.The bottom plate 502 may be a rigid structure to support a cell culture.However, the bottom plate may be a non-solid plate (e.g., may be openand/or porous) to permit oxygen to be provided to the cell culture, asdiscussed in greater detail with reference to FIG. 9. In the illustratedembodiment, the bottom plate 502 is rectangular, or almost rectangular,in shape. In other embodiments, the bottom plate 502 may be any othershape that may enable a low-profile vessel and/or may maximize space inthe location that the first bioreactor vessel may be utilized or stored.

In an embodiment, the vessel body 504 includes a rigid, generallyconcave structure that, when coupled to the bottom plate 502, forms acavity or interior compartment 506 of the first bioreactor vessel 410.As shown therein, the vessel body 504 may have a perimeter shape that issimilar to the perimeter shape of the bottom plate 502 such that thevessel body 504 and the bottom plate 502 may be coupled to one another.Additionally, as in the illustrated embodiment, the vessel body 504 maybe made of a transparent or translucent material that may enable visualinspection of the contents of the first bioreactor vessel 410 and/or mayenable light to enter the first bioreactor vessel 410. The interiorcompartment 506 formed by the bottom plate 502 and the vessel body 504may contain a cell medium and the cell culture during use of the firstbioreactor vessel for cell activation, genetic modification (i.e.,transduction), and/or cell expansion.

As best shown in FIGS. 8-11, the first bioreactor vessel 410 may includemultiple ports through the vessel body 504 that may enable fluidcommunication between the interior compartment 506 and the outside ofthe first bioreactor vessel 410 for certain processes related toactivation, transduction/genetic modification and expansion of cells,such as media input and waste removal. The ports may include, forexample, first port 412 and second port 416. The ports 416 may bedisposed at any location in the vessel body 504, including through a topsurface 508 and/or any of the sides 510 of the vessel body 504, as inthe illustrated embodiment. As will be discussed in greater detailherein, the specific structure of the first bioreactor vessel 410,including the particular quantity and position of the ports 412, 416,enables the first bioreactor vessel 410 to be used to support activationof cells, genetic modification of cells, and high cell densityexpansion.

FIG. 9 is an exploded view of an embodiment of the first bioreactorvessel 410. The bottom plate 502 of the first bioreactor vessel 410 maybe the bottom or support of the first bioreactor vessel 410. Aspreviously discussed, the bottom plate 502 may be formed of a non-solidstructure. In the illustrated embodiment, the bottom plate 502 containsa grid 510 that may be structurally rigid while further providingopening to enable free gas exchange through the bottom plate 502 to theinterior compartment 506 containing the cell culture. The grid 510 mayinclude multiple holes 512 defined between solid areas or crossbars 514between each hole 512 of the grid 510. Thus, the holes 512 may provideopenings for gas exchange, and the crossbars 514 may provide structuralsupport for other structures and the cell culture within the interiorcompartment 506 of the first bioreactor vessel 410.

To provide further support for the cell culture within the interiorcompartment 506 of the first bioreactor vessel 410, the first bioreactorvessel 410 may include a membrane 516 that may be disposed above a topsurface 518 of the bottom plate 502. The membrane 516 may be a gaspermeable, liquid impermeable membrane. The membrane 516 may also beselected having properties enabling high gas permeability, high gastransfer rates, and/or high permeability to oxygen and carbon dioxide.Therefore, the membrane 516 may support high cell densities (e.g., up toabout 35 MM/cm²) within the interior compartment 506. The gaspermeability feature of the membrane 516 may enable the free gasexchange to support the cell culture and/or cell expansion. As such, themembrane 516 may be a cell culture surface and/or cell expansionsurface. The membrane 516 may have a relatively small thickness (e.g.,0.010 inches or 0.02 cm), which may permit the membrane 516 to be gaspermeable. Further, the membrane 516 may be formed from a gas permeablematerial, such as silicone or other gas permeable material.

Flatness of the membrane 516 may increase the surface area for the cellculture to settle on for activation, transduction and/or expansion. Toenable the membrane 516 to remain flat during use of the firstbioreactor vessel 410, a mesh sheet 520 may be disposed between thebottom plate 502 and the membrane 516. The mesh sheet 520 may providestructural support to the membrane 516, such that the membrane 516 mayremain planar and may not sag or distort under the weight of the cellculture and/or any cell medium added to the first bioreactor vessel 410for cell culture and/or cell expansion. Further, the mesh characteristicof the mesh sheet 520 may enable support of the membrane 516, while itsporosity still enables free gas exchange between the interiorcompartment 506 of the first bioreactor vessel 410 and the environmentimmediately outside of the first bioreactor vessel 410. The mesh sheetmay be a polyester mesh, or any other suitable mesh material that mayprovide support to the membrane and enable free gas exchange.

As previously discussed, the vessel body 504 may be coupled to thebottom plate 502 to form the interior compartment 506 of the firstbioreactor vessel 410. As such, the mesh sheet 520 and the membrane 516may be disposed within, or at least partially within, the interiorcompartment 506. An O-ring 522 may be used to seal the first bioreactorvessel 410 when the vessel body 504 is coupled to the bottom plate 502.In an embodiment, the O-ring 522 may be a biocompatible O-Ring (Size173, Soft Viton® Fluoroelastomer O-Ring). The O-ring 522 may fit withina groove 524 formed in a perimetrical surface 526 of the vessel body504. Perimetrical surface 526 faces top surface 518 of plate 502 whenbody 504 is mated to plate 502. As such, the O-ring 522 may becompressed within the groove 524 and against the top surface 518 of theplate 516 and/or the bottom plate 502. Such compression of the O-ring522 desirably seals the first bioreactor vessel 410 without any chemicalor epoxy bonding. As the first bioreactor vessel 410 may be used foractivation, transduction and expansion of biological cells, the O-ring522 is desirably formed from a suitably biocompatible, autoclavable,gamma radiation stable and/or ETO sterilization stable material.

As discussed above, the first bioreactor vessel 410 may include multipleports, such as first port 412 and second port 416. The ports 412, 416may be disposed through the vessel body 504 and may enable communicationbetween the interior compartment 506 and the outside of the firstbioreactor vessel 410 for certain processes related to the cell culture,cell activation, cell transduction, and/or cell expansion, such as fluidor media input, waste removal, collection and sampling. Each port 416may include an opening 526 and a respective fitting or tubing 528 (e.g.,a luer fitting, barb fitting, etc.). In some embodiments, the opening526 may be configured so as to allow for tubing to be bonded directlyand obviate the need for a fitting (e.g., a counterbore).

In an embodiment, in addition to the first port 412 and second port 416,the first bioreactor vessel 410 may further include an air balance port530 disposed in the top surface 508 of the vessel body 504. The airbalance port 530 may be constructed similarly to first port 412 andsecond port 416, where like reference numerals denote like parts. Theair balance port 530 may further provide gas exchange between theinterior compartment 506 and outside of the first bioreactor vessel 410for use by the cell culture for expansion. Further, the air balance port530 may help maintain atmospheric pressure within the interiorcompartment 506 to provide an environment within the interiorcompartment 506 for cell culture and/or cell expansion. The air balanceport 530 may be disposed through the top surface 508 of the vessel body504, as in the illustrated embodiment, or at any other position aboutthe vessel body 504. A central position through the top surface 508 ofthe vessel body 504 may help prevent wetting of the air balance port 530during mixing of the cell culture through tilting of the firstbioreactor vessel 410, as discussed in greater detail below.

Each element of the first bioreactor vessel 410, including the bottomplate 502, the vessel body 504, the ports 412, 416 and 530, the membrane516, the mesh sheet 520, and the O-ring 522, may be made from materialthat are biocompatible, autoclavable, and gamma radiation, and/or ETOsterilization stable. As such, each element, and the first bioreactorvessel 410 as a whole unit, may be used for activation, transduction andexpansion of biological cells, and/or for other processes of the cellmanufacturing process.

The first bioreactor vessel 410 may enable cell culture and/or cellexpansion via perfusion, which may provide nutrients necessary forsupporting cell growth and may reduce impurities in the cell culture.Continuous perfusion is the addition of a fresh media supply to thegrowing cell culture with simultaneous removal of spent media (e.g.,used media). First port 412 and second port 416 may be used for theperfusion process, as discussed below. The first port 412 may enablecommunication between the interior compartment 506 and the outside ofthe first bioreactor vessel 410 and may be used to add a fresh mediuminto the first bioreactor vessel 410 (such as from a culture mediumreservoir of the first fluid assembly 440). In some embodiments, thefirst port 412 may be disposed in and extend through the vessel body 504at any location above the surface of the cell culture and medium withinthe first bioreactor vessel 410. In some embodiments, the first port 412may be disposed such that it contacts or extends through the surface ofthe cell culture and medium within the first bioreactor vessel 410.

The second port 416 may be disposed at any location that is fully orpartially submerged under the surface of the cell culture and the mediumwithin the first bioreactor vessel 410. For example, the second port 416may be a nearly lateral port disposed through one of the sides 510 ofthe vessel body 504. In some embodiments, the second port 416 may bedisposed such that the second port 416 does not reach to the bottom ofthe interior compartment 506 (e.g., the membrane 516). In someembodiments, the second port 416 may reach the bottom of the interiorcompartment 506. The second port 416 may be a dual functionality port.As such, the second port may be used to pull the perfusion media out ofthe interior compartment 506 of the first bioreactor vessel 410 tofacilitate perfusion of the cell culture. Further, the second port 416may also be used to remove the cells of the cell culture. As notedabove, in some embodiments, the second port may not reach the bottomsurface of the interior compartment 506 of the first bioreactor vessel410. For example, the second port 416 may be located approximately 0.5cm away from the membrane 516. Therefore, in a static planar position,the second port 416 may be used to remove the spent cell culture mediumwithout pulling out the cells of the cell culture because the cells maysettle to the membrane 516 (e.g., the cell expansion surface) viagravity. Thus, in the static planar position, the second port 416 mayfacilitate the perfusion process and may enable an increase in the celldensity of the growing cell culture within the first bioreactor vessel410. When cells are desired to be removed from the interior compartment506, for example during harvest of the cell culture, to minimize thehold-up volume, the first bioreactor vessel 410 may be tilted toward thesecond port 416 providing access to the cells for cell removal, in themanner described hereinafter.

Additionally, in an embodiment, the second port 416 may not include afilter and thus, the perfusion process may be filter-free. As such,there may be no physical blockage of the cells from entering the secondport 416 when the second port 416 is used for media removal. Further,the second port 416 may be slanted such that although the second port416 is disposed laterally through the side 22 of the vessel body 504,the second port 416 may be slanted toward the membrane 516 and thebottom plate 502. The slanted feature of the second port 416 may enablethe second port 416 to be positioned relatively low on the vessel body504 closer to the membrane surface 36, while minimizing interferencewith the O-ring 522 and the groove 524 to help maintain sealing of thefirst bioreactor vessel 410 when in use. Further, in some embodiments,the slanted feature of the second port 416 may lower the velocity of thefluid flow through the second port 416 when used medium is removed.Additionally, the port diameter in conjunction with fluid flow rate outof the second port 416 may be such that an inhaling velocity through thesecond port 416 used to pull the media out of the interior compartment506 may minimize suction force on individual cells adjacent to thesecond port 416 such that the force is lower than the gravitationalforce pulling the cells toward the membrane 516. Therefore, as discussedabove, the second port 416 may be used to remove the perfusion medium tofacilitate perfusion of the cell culture without substantially removingthe cells of the cell culture. As the settling time of the cellsincreases, a cell concentration of the removed media may decrease intoan immeasurable range facilitated by the position of the second port416. Further, the position of the interior opening 540 may be changed tochange the recommended cell settling time. Positions closer to themembrane 516 may be associated with longer settling times, whilepositions at or nearer to a top of the medium are associated withshorter settling times, because cells will settle and be first depletedfrom the top of the growth medium.

In an embodiment, the second port 416 may therefore be used not only forremoval of the used media during the perfusion process, but may also beused to remove cells of the cell culture from the interior compartment506, for example during harvest of the cell culture. To facilitategreater removal of the used perfusion medium and removal of cells, thevessel body 504 may include an angled or chevron-shaped sidewall 532.The chevron-shaped sidewall 532 thus includes an apex, or point, 534.Apex 534 of sidewall 532 may further include second port 416therethrough the vessel body 504 is disposed near the point 534 when thevessel body 504 coupled to the bottom plate 502. The angled side 532 andthe point 534 may enable greater drainage of the media and/or the cellsof the cell culture when the first bioreactor vessel 410 is tiltedtoward the second port 416, e.g., at a 5-degree angle.

The use of perfusion to grow the cells facilitated by the positions ofthe first port 412 and the second port 416 may enable a low media height(e.g., 0.3-2.0 cm) within the interior compartment 506, as discussed ingreater detail with reference to FIG. 10. A relatively low media heightwithin the interior compartment 506 may enable the first bioreactorvessel 410 to be a relatively low-profile vessel, while enabling anincrease in the maximum achievable cell density. Further, the use ofperfusion with the first bioreactor vessel 410 may support cell growthby providing fresh medium to the cells within the interior compartment506, but also enable removal of impurities in the cell culture, suchthat additional cell washing in a separate device may not be needed oncea particular cell density goal is reached within the first bioreactorvessel 410. For example, through the filter-free perfusion, the firstbioreactor vessel 410 may provide fresh medium and reduce impuritieswithin the cell culture at a rate of a full volume exchange per day(e.g., resulting in an impurity reduction at a rate of approximately 1log per 2.3 days). Therefore, the structure of the first bioreactorvessel 410 may enable the use of perfusion for growing the cell culturewithin the first bioreactor vessel 410, which may thus enable expansionof the cell culture to a high target density with a reduced impuritylevel. As also discussed hereinafter, through the filter-free perfusion,the first bioreactor vessel 410 may provide fresh medium at a rate of asubstantially more volumes per day (e.g., greater than 2 volumes perday) for seeding, rinsing, washing/residual reduction, and/ordraining/harvesting of the cells after expansion.

To facilitate a low-profile structure of the first bioreactor vessel410, a relatively low media height within the interior compartment 506may be maintained. FIG. 10 is a cross-sectional view of the firstbioreactor vessel 410 illustrating a height 536 of cell media 538 withinthe first bioreactor vessel 410. As previously discussed, the vesselbody 504 may be coupled to the bottom plate 502 to form the interiorcompartment 506 within which expansion of the cell culture may beachieved through perfusion. As such, replacement or fresh medium 538 maybe provided for cell growth through the first port 412 disposed throughthe vessel body 504, and existing or used medium 538 may be removedthrough the second port 416 disposed through the side 510 of the vesselbody 504. The perfusion process may facilitate relatively low mediumheight 536 of the medium 538 within the interior compartment 506 of thefirst bioreactor vessel 410. The relatively low height 536 of theperfusion medium 538 within the interior compartment 506 may enable thefirst bioreactor vessel 410 to be a low-profile structure, which thus,may enable a compact cell manufacturing system as a whole.

The height 536 of the perfusion medium 538 within the interiorcompartment 506 of the first bioreactor vessel 410 may be between 0.3 cmand 2 cm, and the height of the head room 542, i.e., a gap formedbetween the medium 538 and the top surface 508 of the vessel body 504 inthe interior compartment 506, may be approximately 2 cm. Thus, there maybe less than 2 mL of media per cm² and less than 4 mL of total volumeper cm², including the media, the cell culture, and headspace. Arelatively low media height 536 may enable a ratio of media volume tosurface area of the membrane 516 to be below a certain value. As such,the ratio of the medium volume to the membrane surface area may be belowa threshold level, or within a desirable range, facilitated by the useof perfusion to grow the cells of the cell culture. For example, thethreshold level may be a ratio between 0.3-2.0. The low ratio mediumvolume to membrane surface area may enable the first bioreactor vessel410 to have a low-profile or compact structure, while still permitting ahigh cell density cell culture to be achieved.

As previously discussed, the dual functionality second port 416 may bedisposed through the vessel body 504 such that it is fully or partiallysubmerged under a surface 544 of the medium 538 within the firstbioreactor vessel 410. In some embodiments, the second port 416 may bedisposed such that the second port 416 reaches to the bottom of theinterior compartment 506 (e.g., the membrane 516). Positioning of thesecond port 416 may facilitate media and impurity removal from the cellculture within the interior compartment 506, without removal of thecells until such removal is desired, for example harvesting. Thefilter-free second port 416, along with the first port 412, may permitthe use of perfusion to provide the growth medium 538 to the cells forcell expansion, and to remove the used medium 538 and other impuritiesor byproducts. The position of the first port 412 and the dualfunctioning second port 416 about the vessel body 504 facilitates aconfiguration in which the height 536 of the medium within the interiorcompartment 506 to be maintained at a relatively low level and thus,permit the first bioreactor vessel 410 to be a relatively low-profilevessel, while still permitting generation of a high-density cellculture.

With specific reference to FIG. 11, the bottom plate 502 of thebioreactor vessel 410 includes a variety of features that enable use ofthe bioreactor vessel as part of the broader bioprocessing system 10 andin particular, the second module 200 of the bioprocessing system 10. Asshown therein, the bottom plate 502 includes a plurality of recesses 550formed in a bottom surface of the bottom plate 502, the purpose of whichwill be described hereinafter. In an embodiment, the recesses may belocated adjacent to the corners of the bottom plate 502. The recesses550 may each be generally cylindrical in shape and terminate at adome-like or hemispherical-like interior surface. As also shown in FIG.11, the bottom plate 502 may include a position verification structure552 that is configured to interact with a sensor of the second module200 to ensure proper positioning of the first bioreactor vessel 410within the second module 200. In an embodiment, the positionverification structure may be a beam break that is configured tointerrupt an optical beam of the second module 200 when the firstbioreactor vessel 410 is properly seated therein.

The bottom plate 502 also includes a pair of flat engagement surfaces554 formed on the bottom surface adjacent, which are offset from acenter line of the bottom plate (that extends across the width of thebottom plate). Desirably, the engagement surfaces 554 are spaced-apartalong a longitudinal centerline of bottom plate 502 so as to bepositioned adjacent to opposed ends of the bottom plate 502. The bottomplate 502 may further include at least one aperture or opening 556 toallow for sensing of the contents of the first bioreactor vessel 410 bya bioprocessing apparatus which engages and operates the bioreactorvessel.

In an embodiment, the first and second bioreactor vessels 410, 420 andthe fluid architecture 400 may be integrated into an assembly or kit 600in the manner disclosed below. In an embodiment, the kit 600 is asingle-use, disposable kit. As best shown in FIGS. 12-14, the firstbioprocessing vessel 410 and the second bioprocessing vessel 420 arereceived side-by-side within a tray 610 of the disposable kit 600, andthe various tubes of the flow architecture 400 arranged within the tray610 in the manner described hereinafter.

With additional reference to FIG. 15, the tray 610 includes a pluralityof generally thin, rigid or semi-rigid sidewalls including a front wall612, a rear wall 614, and opposed lateral sides 616, 618 perimetricallybounding a bottom surface 620 and a generally open top. The sidewallsand bottom surface 620 define an interior compartment 622 of the tray610. In an embodiment, the open top of the tray 610 is bounded by aperipheral flange 624 that presents a surface for receiving removablecover (not shown) that encloses the interior compartment 622 as well asfor desirably seating on an upper rim of a drawer of a bioprocessingapparatus, as indicated below. The bottom surface 620 of the tray 610includes a number of openings corresponding to the number of bioreactorvessels in the bioprocessing system. For example, the tray 610 mayinclude a first opening 626 and a second opening 628. The bottom surface620 may also include an additional opening 630 adjacent to the first andsecond openings 626, 628 for the purpose described below. In anembodiment, the tray 610 may be thermoformed, 3D printed, or injectionmolded, although other manufacturing techniques and processes may alsobe utilized without departing from the broader-aspects of the invention.

As best shown in FIG. 15, each of the first and second opening 626, 628has a perimeter that is shaped and/or dimensioned such that the firstand second bioreactor vessels 410, 420 can be positioned above therespective openings 626, 628 and supported by the bottom surface 620 ofthe tray 610 within the interior compartment 622, while still allowingfor a portion of the bioreactor vessels 610, 620 to accessible from thebottom of the tray 610 through the respective openings 626, 628. In anembodiment, the perimeter of the openings include at least one tab orprojection for supporting the bioreactor vessels above the respectiveopenings. For example, the perimeter of each opening 626, 628 mayinclude tabs 632 that project inwardly towards the center of theopenings 626, 628 for supporting the bioreactor vessels 410, 420 placedthereon. As shown in FIGS. 12 and 15, the tray 610 may also include oneor more bosses extending upwardly above the openings 626, 628 forinhibiting lateral movement of the bioreactor vessels when they arereceived above the respective openings 626, 628. The bosses thereforeserve as alignment devices that facilitate proper positioning of thebioreactor vessels 410, 420 within the tray 610, and help to preventinadvertent movement of the bioreactor vessels 410, 420 during loadingor positioning of the kit 600 in the second module 200, as discussedbelow.

With further reference to FIGS. 12 and 13, the tray 610 may include oneor more support ribs 636 formed on the bottom surface of the tray 610.The support ribs 636 may extend across the width and/or length of thetray 610 and impart rigidity and strength to the tray 610, facilitatingmovement and manipulation of the kit 600. The ribs 636 may be integrallyformed with the tray or may be added as an auxiliary component viaattachment means known in the art. (See FIG. 13). In an embodiment, thetray 610 includes an opening 638 for receiving an engagement plate, alsoreferred to herein as tubing module 650, therethrough, which retains thefluid flow lines in an organized manner and holds them in position forengagement by the pumps and pinch valves. In other embodiments, thetubing module 650 may be integrally formed with the rear wall 614 of thetray 610.

FIGS. 16 and 17 illustrate the configuration of the tubing module 650according to an embodiment of the invention. As shown therein, thetubing module 650 includes a first tubing holder block 652 configured toreceive the first fluid assembly line 442, the interconnect line 450 andthe waste line 490 of the fluid flow system 400, and hold the firstfluid assembly line 442, the interconnect line 450 and the permeatewaste line 490 in position for selective engagement with respective pumpheads 454, 456, 492 of a peristaltic pump assembly described below inconnection with FIGS. 35 and 36. In an embodiment, the fluid assemblyline 442, interconnect line 450 and waste line 490 are maintained inhorizontally-extending and vertically-spaced orientation by the firsttubing holder block 652. In particular, as best shown in FIG. 17, thefirst tubing holder block 652 engages each the lines 442, 450, 490 attwo spaced-apart locations 656, 658 (such as through clips or simpleinterference between the tubes and slots in the tubing holder block 652)that define a void therebetween. As also shown in FIG. 17, the firsttubing holder block 652 includes a clearance opening 660 that isconfigured to receive a shoe (not shown) of the peristaltic pumpassembly. This configuration allows for peristaltic compression of thelines 442, 450, 490 against the shoe by the respective pump heads of theperistaltic pump(s) so as to provide a respective motive force of fluidthrough the lines, as discussed below.

With further reference to FIGS. 16-18, the tubing module 650 furtherincludes a second tubing holder block 654 integrally formed with (orotherwise coupled to) the first tubing holder block 652. The secondtubing holder block 654 is configured to receive all of the fluid flowlines of the fluid flow system 400 with which pinch valves areassociated. For example, the second tubing holder block 654 isconfigured to retain the tubing tails 464 a-f of the first fluidassembly 440, the tubing tails 470 a-d of the second fluid assembly 444,the first bioreactor line 414 and second bioreactor line 418 of thefirst bioreactor vessel 410, the first bioreactor line 424 and thesecond bioreactor line 428 of the second bioreactor vessel 420, thesterile air source line 460, the interconnect line 450 and thefiltration line 482 (and in some embodiment, the sampling lines 476a-476 d). Similar to the first tubing holder block 652, the secondtubing holder block 654 may maintain these tubes inhorizontally-extending and vertically-spaced orientation. In particular,the second tubing holder block 654 may include a plurality orvertically-spaced and horizontally-extending slots 666 that areconfigured to receive the lines therein. FIGS. 18 and 19 also bestillustrate the configuration of the slots 666 that retain all of theflow lines that are acted upon by/interface with the pinch valves.Desirably, slots 666 follow the contour of block 654 but particularlyextends across planar back plate so as to open towards filter 484. Asshown in FIG. 18, in an embodiment, the second tubing holder block 654may have one or more narrow tubing slots 682 at the bottom of the secondtubing holder block 654 for holding a loop of the interconnect line 450,from which the sampling lines extend, and a waste line tubing slot 684for receiving the tubing tail 470 a that is connected to the wastereservoir 472 a.

The second tubing holder block 654 may include a planar back plate 662having a plurality of apertures 664 corresponding to the plurality offluid flow lines retained by the second tubing holder block 654. Inparticular, at least one aperture 664 is horizontally aligned with eachslot 666 and flow line retained therein. As best shown in FIG. 16, thesecond tubing holder block 654 includes two clearance openings 668, 670that are configured to receive an anvil (not shown) of a pinch valveassembly therethrough. This configuration allows for selectivecompression of the tubing tails 464 a-f of the first fluid assembly 440,the tubing tails 470 a-d of the second fluid assembly 444, the firstbioreactor line 414 and second bioreactor line 418 of the firstbioreactor vessel 410, the first bioreactor line 424 and the secondbioreactor line 428 of the second bioreactor vessel 420, the sterile airsource line 460, the interconnect line 450 and the filtration line 482against the anvil by a respective piston of an actuator of the pinchvalve array, to selectively prevent or allow fluid flow, as discussedbelow. As shown in FIGS. 18 and 19, the apertures 664 may be arranged infirst and second columns positioned side by side, wherein the aperturesin the first column of apertures are offset in a vertical direction withrespect to the apertures on the second column of apertures so that theapertures in the first column of apertures are not in horizontalalignment with the apertures in the second column of apertures. Thisconfiguration allows for the tubing module 650, tray 610 and kit 600, asa whole, to have a low profile.

In an embodiment, the filter 484 (shown in FIG. 16 as an elongate hollowfiber filter module) may be integrated with the tubing module 650, suchas by mounting the filter 484 to the tubing module 650 through the useof retaining clips 672. Where the filter 484 is a hollow fiber filter,the filter 484 may extend substantially the entire length of the tubingmodule 650 and may include a first, input end 674 for receiving an inputflow of fluid from the filtration line 482, and a second, output end 676for conveying the retentate, after removal of permeate/waste, back tothe filtration line 482 and interconnect line 450 for circulation to oneof the first bioreactor vessel 410 or second bioreactor vessel 420. Thefilter 484 may also include a permeate port 678 adjacent to the second,output end 676 for connection to the waste line 490 for conveying thewaste/permeate to permeate/waste reservoir 472 a. Finally, the tubingmodule 650 may include a plurality of features 680 for receiving clipsand organizing the bioreactor lines (e.g., first and second bioreactorlines 414, 418 of the first bioreactor vessel 410 and/or first andsecond bioreactor lines 424, 428 of the second bioreactor vessel 420).

Similar to the tray 610, the tubing module 650 may be thermoformed, 3Dprinted, or injection molded, although other manufacturing techniquesand processes may also be utilized without departing from thebroader-aspects of the invention. As discussed above, in an embodiment,the tubing module 650 may be integrally formed with the tray 610. Inother embodiments, the tubing module 650 may be a separate componentthat is removably received by the tray 610.

FIGS. 20-22 show various views of an embodiment of the kit 600,illustrating the first bioreactor vessel 410 and the second bioreactorvessel 420 received within the tray 610 and the fluid lines of the flowarchitecture 400 received by the tubing module 650. As shown therein,rather than having an opening 630, the kit 600 as shown in FIGS. 20-22includes a solid floor there so as to provide a sampling space 631 intray 610 for receiving a container that holds the sampling lines (e.g.,sampling lines 476 a, 476 b). The kit 600 provides for a modularplatform for cell processing that can be easily set up and discardedafter use. The tubing tails of the first and second fluid assemblies440, 444 allow for plug-and-play functionality, enabling the quick andeasy connection of various media, reagent, waste, sampling andcollection bags to allow for a variety of processes using to be carriedout on a single platform. In an embodiment, connection and disconnectioncan accomplished by sterile cutting and welding of tube segments, asdiscussed above, such as with a TERUMO device, or by pinching, welding,and cutting the tail segment as is known in the art.

Turning now to FIGS. 23-25, the kit 600 is specifically configured to bereceived by a bioprocessing apparatus 700 that contains all of thehardware (i.e., controllers, pumps, pinch valve actuators, etc.)required for actuating kit 600 as part of a bioprocessing method. In anembodiment, the bioprocessing apparatus 700 and kit 600 (containing theflow architecture 400 and bioreactor vessels 410, 420) together form thesecond bioprocessing module 200 described above in connection with FIGS.1 and 2. The bioprocessing apparatus 700 includes a housing 710 having aplurality of drawers 712, 714, 716 receivable within the housing 710.While FIG. 23 depicts an apparatus 700 containing three drawers, theapparatus may have as few as a single drawer, two drawers, or more thanthree drawers to provide for simultaneous bioprocessing operations to becarried out within each drawer. In particular, in an embodiment, eachdrawer 712, 714, 716 may be a stand-alone bioprocessing module forcarrying out the processes of cell activation, genetic modificationand/or expansion (i.e., equivalent to the second modules 200 a, 200 band 200 c described above in connection with FIG. 2). In this respect,any number of drawers may be added to the apparatus 700 to provide forparallel processing of multiple samples from the same or differentpatients. In an embodiment, rather than each drawer sharing a commonhousing, in an embodiment, each drawer may be received within adedicated housing, and the housings can be stacked atop one another.

As shown in FIGS. 23 and 24, each drawer, e.g. drawer 712, includes aplurality of sidewalls 718 and a bottom surface 720 defining aprocessing chamber 722, and a generally open top. The drawer 712 ismovable between a closed position in which the drawer is fully receivedwithin the housing 710, as shown for drawers 714 and 716 in FIG. 23, andan open position, as shown for drawer 712 in FIGS. 23 and 24, in whichthe drawer 712 extends from the housing 710 enabling access to theprocessing chamber 722 through the open top. In an embodiment, one ormore of the sidewalls 718 are temperature-controlled for controlling atemperature within the processing chamber 722. For example, one or moreof the sidewalls 718 may include an embedded heating element (notshown), or be in thermal communication with a heating element, so thatthe sidewalls 718 and/or processing chamber 722 may be heated to adesired temperature for maintaining the processing chamber 722 at adesired temperature (e.g., 37 degrees Celsius) as optimized for processsteps to be performed by module 200. In some embodiments, the bottomsurface 720 and the underside of the top surface of the housing (abovethe processing chamber when the drawer is closed) may betemperature-controlled in a similar manner (e.g., an embedded heatingelement). A hardware compartment 724 of the drawer 712 behind theprocessing chamber 722 may house all of the hardware components of theapparatus 700, as discussed in detail hereinafter. In an embodiment, thedrawer 712 may further include an auxiliary compartment 730 adjacent tothe processing chamber 722 for housing the reservoirs containing media,reagents, etc. that are connected to the first fluid assembly 440 andsecond fluid assembly 444. In an embodiment, the auxiliary compartment730 may be refrigerated.

Each drawer, e.g., drawer 712, may be slidably received on opposed guiderails 726 mounted to the interior of the housing 710. A linear actuatormay be operatively connected to the drawer 712 to selectively move thedrawer 712 between the open and closed positions. The linear actuator isoperable to provide smooth and controlled movement of the drawer 712between the open and closed positions. In particular, the linearactuator is configured to open and close the drawer 712 at asubstantially constant speed (and minimal acceleration and decelerationat the stop and start of the motion) to minimize disturbance to thecontents of the bioreactor vessels(s).

FIG. 25 is a top plan view of the interior of the drawer showing theprocessing chamber 722, the hardware compartment 724 and the auxiliarycompartment 730 of the drawer 712. As illustrated therein, the hardwarecompartment 724 is located rearward of the processing chamber 722includes a power supply 732, a motion control board and driveelectronics 734 that are integrated with or otherwise in communicationwith the second module controller 210, a low-power solenoid array 736,the pump assembly 738 (which includes pump heads for the pumps 454, 456,492) and a drawer engagement actuator 740. The hardware compartment 724of the drawer 712 further includes a pump shoe 742 and a pair of pinchvalve anvils 744 for interfacing with the pump assembly 738 and thesolenoid array 736, respectively, as described hereinafter. In anembodiment, the pump shoe 742 and the solenoid anvil 744 are fixed tothe front base plate of the processing chamber (the front plate). Thehardware compartment (and the components described) are all mounted tothe back base plate. Both the plates are slidably mounted to the rails.Further, the drawer engagement actuator 740 couples the two plates andis used to bring the two plates (and the components carried on theplates to an engagement position (bringing the pump roller heads intothe pump shoe and thereby squeezing the pump tubing if insertedbetween). As is further described herein, pump assembly providesselective operation on lines 442, 450 and 490 of fluidpath 400 toprovide independent respective peristaltic motive forces therefor.Similarly, tubing holder block 654 of tray 600 will be positionedbetween the solenoid array 736 and the anvils 744 as will be describedfurther.

As also illustrated in FIG. 25, two bed plates, e.g., first and secondbed plates 746, 748, are located within the processing chamber 722 onthe bottom surface 720 and extend upwardly or stand proud therefrom. Inan embodiment, the processing chamber 722 may house a single bed plate,or more than two bed plates. The bed plates 746, 748 are configured toreceive or otherwise engage the first bioreactor vessel 410 and secondbioreactor vessel 420 thereon. As also shown in FIG. 25, the drawer 712also includes a plate 750 configured with load cells positioned adjacentto the bed plates 746, 748 within the processing chamber 722 for sensinga weight of a reservoir, e.g., waste reservoir 472 a positioned thereon.

FIGS. 26-28 best illustrate the configuration of the bed plates 746,748, with FIG. 28A showing the hardware components positioned beneaththe bed plate. A used herein, the bed plates 746, 748 and the hardwarecomponents (i.e., sensors, motors, actuators, etc. integrated therewithor positioned therebeneath as shown in FIG. 28A) may collectively bereferred to as the bed plate. The first and second bed plates 746, 748are substantially identical in configuration and operation, but forsimplicity, the following description of the bed plates 746, 748 makesreferences only to the first bed plate 746. The bed plates 746, 748 havea substantially planar top surface 752 having a shape and surface areathat generally corresponds to the shape and area of the bottom plate 502of the first bioreactor vessel 410. For example, the bed plate may begenerally rectangular in shape. The bed plates 746, 748 may also includerelief or clearance areas 758, that generally correspond to the positionof the projections or tabs 632 of the tray 610, the purpose of whichwill be described below. The bed plates 746, 748 are supported by aplurality of load cells 760 (e.g., four load cells 760 positionedbeneath each corner of the bed plate 746). The load cells 760 areconfigured to sense the weight of the first bioreactor vessel 410 duringbioprocessing, for use by the controller 210.

In an embodiment, the bed plate 746 may include an embedded heatingelement or be in thermal communication with a heating element so thatthe processing chamber 722 and/or the contents of the first bioreactorvessel 410 placed thereon can be maintained at a desired temperature. Inan embodiment, the heating element may be the same or different than theheating element that heats the sidewalls 718, top wall and bottomsurface.

As illustrated, the bed plate 746 includes plurality of locating oralignment pins 754 that protrude above the top surface 452 of the bedplate 746. The number of locating pins 754 and the position and spacingof the locating pins 754 may correspond to the number, position andspacing of the recesses 550 in the bottom surface of the bottom plate502 of the bioreactor vessels 410, 420. As indicated below, the locatingpins 754 are receivable within the recesses 550 in the bottom plate 502of the first bioreactor vessel 410 when the first bioreactor vessel 410is positioned within the processing chamber 722 to ensure properalignment of the first bioreactor vessel 410 on the first bed plate 746.

With further reference to FIGS. 26-28, the bed plate 746 may furtherinclude an integrated sensor 756 for detecting proper alignment (ormisalignment) of the first bioreactor vessel 410 on the first bed plate746. In an embodiment, the sensor 756 is an infrared optical beam,although other sensor types such as a lever switch may also be utilizedwithout departing from the broader aspects of the invention. The sensoris configured to interact with the position verification structure 552on the bottom plate 502 when the first bioreactor vessel 410 is properlyseated on the first bed plate 746. For example, where the sensor 756 isan infrared optical beam and the position verification structure 552 isa beam break (i.e., a flat tab), with a substantially IR-opaque positionverification structure 552, when the first bioreactor vessel 410 isfully seated on the bed plate 746, the beam break will interrupt theinfrared optical beam (i.e., break the beam). This will signal to thecontroller 210 that the first bioreactor vessel 410 is properly seated.If, after positioning the first bioreactor vessel 410 on the first bedplate 746, the controller does not detect that the infrared optical beamof the sensor 756 is broken, this indicates that the first bioreactorvessel 410 is not fully or properly seated on the bed plate 746 and thatadjustment is needed. The sensor 756 on the bed plate 746 and positionverification structure 552 on the bottom plate 502 of the firstbioreactor vessel 410 therefore ensure that the first bioreactor vessel410 is seated in level position on the bed plate 746 (as determined bythe alignment pins) prior to commencing bioprocessing.

Referring still further to FIGS. 26-28A, the bed plate 746 additionallyincludes an embedded temperature sensor 759 that is positioned so as tobe in alignment with the aperture 556 in the bottom plate 502 of thefirst bioreactor vessel 410. The temperature sensor 759 is configured tomeasure or sense one or more parameters within the bioreactor vessel 410such as, for example, a temperature level within the bioreactor vessel410. In an embodiment, the bed plate 746 may additionally include aresistance temperature detector 760 configured to measure a temperatureof the top surface 752, and a carbon dioxide sensor (located under thebed plate) for measuring a carbon dioxide level within the bioreactorvessel.

As further shown in FIGS. 26-28A, each bed plate 746, 748 includes anactuator mechanism 761 (e.g., a motor) that includes, for example, apair of opposed cam arms 762. The cam arms 762 are received within slots764 in the bed plates 746, 748, and are rotatable about cam pin 766between a clearance position where the cam arms 762 are positionedbeneath the top surface 752 of the bed plate 746, and an engagementposition where the cam arms 762 extend above the top surface 752 of thebed plate and contact the opposed flat engagement surfaces 554 of thebottom plate 502 of the first bioreactor vessel 410 when the firstbioreactor vessel 410 is received atop the first bed plate 746. Asdiscussed in detail below, the actuator mechanism is operable to tiltthe bioreactor vessel atop the bed plate to provide for agitation and/orto assist draining of the bioreactor vessel.

Referring to FIGS. 29-32, more detailed views of the linear actuator 768and drawer engagement actuator 740 in the hardware compartment 724 ofthe drawer 712 are shown. With reference to FIG. 29, and as indicatedabove, the linear actuator 768 is operable to move the drawer 712between the open and closed positions. In an embodiment, the linearactuator 768 is electrically connected to a rocker switch 770 on theexterior of the housing 710 which allows for user control of themovement of the drawer. The linear actuator 770 provides controlledmovement of the drawer 712 to prevent disturbance of the contents of thebioreactor vessel(s) within the drawer 712. In an embodiment, the linearactuator 768 has a stroke of approximately 16″ and has a maximum speedof approximately 2 inches per second.

Turning now to FIG. 30, the drawer engagement actuator 740 includes alead screw 772 and a clevis arm 774 that attaches to a front plate 751within the drawer 712. The drawer engagement actuator is operativelyconnected to the pump assembly 738 and the solenoid array 736 and isoperable to move the pump assembly 738 and the solenoid array 736between a first, clearance position and an engagement position.

FIGS. 31 and 32 better illustrate the clearance position and engagementposition of the pump assembly 738 and solenoid array 736. As illustratedin FIG. 31, in the clearance position, the pump assembly 738 andsolenoid array 736 are spaced from the pump shoe 742 and pinch valveanvils 744, respectively. Upon actuation of the lead screw 772, thedrawer engagement mechanism 740 moves the pump assembly 738 and solenoidarray linearly forward to the position shown in FIG. 32. In thisposition, the pump heads of the pump assembly 738 engage the lines 442,450, 490 in the first tubing holder block 652 and the solenoid array 736is positioned close enough to the pinch valve anvils 744 that apiston/actuator of the solenoid array 736 can pinch/clamp its respectivefluid flow lines of the second tubing holder block 654 against the pinchvalve anvil(s) 744, thereby preventing flow through that fluid flowline.

Referring back to FIG. 24, and with additional reference to FIGS. 33-39,in operation, the drawer 712 may be controllably moved to the openposition by actuating the rocker switch 770 on the outside of thehousing 710. The disposable drop in kit 600 containing the tubing module650 (which holds all the tubes and tubing tails of the flow architecture400) and first and second bioreactor vessels 410, 420 is then loweredinto position within the processing chamber 722. As the kit 600 islowered into the processing chamber 722, the pump shoe 742 is receivedthrough the clearance opening 660 of the first tubing holder block 652so that the pump tubes 442, 450, 490 are positioned between the pumpshoe 742 and the pump heads 454, 456, 492 of the peristaltic pumpassembly 738. FIG. 35 is a perspective view of the peristaltic pumpassembly 738, showing the positioning of the pump heads 454, 456, 492 inrelation to one another. FIG. 36 illustrates the positioning of the pumpheads 454, 456, 492 in relation to the pump tubes 442, 450, 490 when thekit 600 is received within the processing chamber 722. As shown therein,the pump tubes 442, 450, 490 are positioned between the pump shoe 742and the pump heads 454, 456, 492. In operation, when the drawerengagement actuator 740 positions the pump assembly 738 in theengagement position, the pump heads 454, 456, 492 are selectivelyactuatable under control of the controller 210 to initiate, maintain andcease a flow of fluid through the tubes 442, 450, 490.

Similarly, as the kit 600 is lowered into the processing chamber 722,the pinch valve anvils 744 are received through the clearance openings668, 670 of the second tubing holder block 654 so that the tubing tails464 a-f of the first fluid assembly 440, the tubing tails 470 a-d of thesecond fluid assembly 444, the first bioreactor line 414 and secondbioreactor line 418 of the first bioreactor vessel 410, the firstbioreactor line 424 and the second bioreactor line 428 of the secondbioreactor vessel 420, the sterile air source line 460, the interconnectline 450 and the filtration line 482 that are retained by the secondtubing holder block 654 are positioned between the solenoid array 736and the pinch valve anvils 744. This configuration is best illustratedin FIGS. 37-39 (FIGS. 37 and 38 illustrating the relationship betweenthe solenoid array 736 and the pinch valve anvils 744 prior to receivingthe back plate 662 of the second tubing holder block 654 within space776).

As shown therein, each solenoid 778 of the solenoid array 736 includes apiston 780 that is extendable linearly through an associated aperture(of apertures 664) in the back plate 662 of the second tubing holderblock 654 to clamp an associated tube against the pinch valve anvil 744.In this respect, the solenoid array 736 and the anvil 744 together forma pinch valve array (which includes the valves of the first fluidassembly 440 and second fluid assembly 444, as well as the bioreactorline valves, i.e., valves 432, 434, 436, 438, sterile line valve 462,interconnect line valve 452 and filtration line valves 486, 488). Inparticular, the pinch valves of the flow architecture 400 are providedby the respective solenoids 778 (i.e., pistons of the solenoids) of thesolenoid array 736 operating/acting against its respective anvil 744while the fluid path/line is in between. In particular, in operation,when the drawer engagement actuator 740 positions the solenoid array 736in the engagement position, each solenoid 778 is selectively actuatableunder control of the controller 210 to clamp an associated fluid flowline against the anvil 744 to prevent a flow of fluid therethrough. Thepresent invention contemplates that each fluid line is positionedbetween a planar anvil face and a planar solenoid actuator head.Alternatively, the solenoid actuator head may include a shaped head,such as a two tapering surfaces meeting at an elongate edge akin to aPhillips-head screwdriver, that is optimized to provide a desiredpinching force on the resiliently-flexible fluid line. Alternativelystill, the anvil face may include an elongate ridge or projectionextending towards each fluid line such that a planar solenoid head maycompress the fluid line against this transversely-extending ridge so asto close the line to fluid flow therethrough.

With reference to FIGS. 33, 34 and 40, as the kit 600 is lowered intothe processing chamber of the drawer, the first bioreactor vessel 410and the second bioreactor vessel 420 are supported above the openings626, 628 by the perimeter of the openings and, in particular, by thetabs/projections 632. As the kit is lowered further, the bed plates 746,748 extend through the openings 626, 628 and receive or otherwise engagethe bioreactor vessels 410, 420. The shape of the openings 626, 628 andthe top surface 752 of the bed plates 746, 748 (e.g., relieved areas 758of the bed plates 746, 748 that correspond to the tabs/projections 632of the tray 610) allow the tray 610 to continue downward travel once thebioreactor vessels 410, 420 are received by the bed plates 746, 748 suchthat the bottom surface of the tray 610 and the tabs/projections 632 areseated at a location lower than the top surface 752 of the bed plates746, 748 so that the bioreactor vessels 410, 420 can be supported by thebed plates 746, 748 in spaced relationship to the bottom surface 620 ofthe tray 610. This ensures that the tray 610 does not interfere with thelevel seating of the bioreactor vessels 410, 420 on the bed plates 746,748.

As the bed plates 746, 748 extend through the openings 726, 728 in thetray 610, the locating pins 754 on the bed plates 746, 748 are receivedin the corresponding recesses 550 in the bottom plate 502 of thebioreactor vessels 410, 420, ensuring that the bioreactor vessels 410,420 will be properly aligned with the bed plates 410, 420. When properlyseated on the bed plates 746, 748, the beam break 552 breaks the opticalbeam of the sensor 756 in the bed plates, indicating to the controllerthat the bioreactor vessels 410, 420 are in proper position. Because thebed plates 746, 748 and the alignment pin heights are level,interruption of the optical beam of the sensor 756 by the beam break 552likewise ensures that the bioreactor vessels 410, 420 are level. In thisproperly seated position, sensor 759 on the bed plates 746, 748 isaligned with the aperture 556 in the bottom plate 502 to allow forsensing of processing parameters within the interior compartment of thebioreactor vessels 410, 420, respectively. In addition, in the fullyseated position, the cam arms 762 of the bed plates 746, 748 are alignedwith the flat engagement surfaces 554 on the bottom plate 502 of thebioreactor vessels 410, 420, respectively.

FIG. 40 is a cross-sectional, front view illustrating this fully seatedposition of the first bioreactor vessel 410 on the bed plate 746. Asshown in FIG. 40, a heating element in the form of a heating pad 782 andheating module 784 may be positioned below the bed plate 746 for heatingthe bed plate 746. As shown in FIG. 40, a carbon dioxide sensing module786 may also be positioned beneath the bed plate for sensing a carbondioxide content within the processing chamber 722.

As further shown in FIG. 40, in an embodiment, the sidewalls 718 andbottom, of the drawer 712 (and the top wall of the housing) may comprisea cover 788, an insulative foam layer 790 to help minimize heat lossfrom the processing chamber 722, a film heater 792 for heating the wallsas described above, and an inner metal plate 794. In an embodiment, theinner metal plate 794 may be formed from aluminum, although otherthermally conductive materials may also be utilized without departingfrom the broader aspects of the invention. The drawer 712 may furtherinclude one or more brush seals 796 to help minimize heat loss from theprocessing chamber 722, and a thermal break 798 to minimize or preventthe flow of thermal energy from the drawer 712 to other components ofthe apparatus 700 (such as housing 710 or other drawers (e.g., drawers714, 716)).

Referring once again to FIG. 34, when the kit 600 is received in theprocessing chamber 722, the load cell 750 in the bottom of theprocessing chamber 722 adjacent to the second bed plate 748 extendsthrough the opening 730 in the tray 610 so that a waste bag 472 a may beconnected to the tubing tail 470 a and positioned on the load cell 750.As shown therein, when the kit 600 is received within the drawer 712,the second tubing holder block 654 retains the tubing such that thetubing tails 464 a-f of the first fluid assembly 440 and the tubingtails 470 b-d of the second fluid assembly 444 extend into the auxiliarycompartment 730 for the connection of the reservoirs thereto. In anembodiment, the sampling lines 476 a-476 d likewise extend into theauxiliary compartment 730.

Turning now to FIGS. 41-44, operation of the cam arms 762 of the bedplates 746, 748 is illustrated. As shown therein, the cam arms 762 aremovable between a retracted position where they are positioned beneaththe top surface of the bed plates 746, 748 and an engagement positionwhere they are rotated about cam pin 766 and extend above the bed plates746, 748 to engage the flat engagement surfaces 554 of the bioreactorvessels 410, 420 to lift the bioreactor vessels 410, 420 off of the bedplates 746, 748. Because the cam arms 762 are retracted beneath the topsurface of the bed plates 746, 748 in a default state and the bioreactorvessels 410, 420 are supported on the level bed plates 746, 748 (and,particularly, the level alignment pins 754, no power is needed tomaintain the bioreactor vessels in a level position. In particular, whenthe bioreactor vessels 410, 420 are received on the bed plates 746, 748,they are in level position. In the event of a power interruption, thebioreactor vessels 410, 420 remain seated on the level bed plates 746,748 and do not require any continual adjustment using the cam arms 762to maintain the level position. This is in contrast to some systemswhich may require constant adjustment of the bioreactor usingservomotors to maintain a level position. Indeed, with configuration ofthe cam arms 762 of the invention, the actuator need only be energizedwhen tilting the bioreactor vessels for agitation/mixing, as discussedbelow, which minimizes heat contribution to the processing chamber 722.

As shown in FIGS. 41-43, the cam arms 762 may be operable sequentiallyto agitate the contents of the bioreactor vessels 410, 420. For example,when it is desired to agitate the contents of the bioreactor vessel 410,one of the cam arms will be actuated to lift one end of the bioreactorvessel 410 off of the bed plate 746 (and out of engagement with thelocating pins 754 on the bed plate 746, while the opposing end remainsseated on the bed plate and the locating pins 754 on the non-raised endremain received in the corresponding recesses 550 in the bottom plate502. The raised cam arm will then be rotated back to the clearanceposition beneath the bed plate and the opposing cam arm will be rotatedto the engagement position to raise the opposing end of the bioreactorvessel off of the bed plate and locating pins.

In an embodiment, the cam actuation system may be designed such that thecam arms 762 can be homed without touching the bioreactor vessel,preventing disruption to the culture and allowing the cam arms 762 to behomed (or tested) at any point during the long cell processing periods.Thus while the present invention contemplates that other rocking oragitations means may be provided for the bioreactor vessels, by havingtwo cam arms 762 on opposite sides of the bed plate, the overall heightof the mixing mechanism can be minimized. For example a +/−5-degreemotion could be achieved with a central actuator (located centrally onthe bed plate), but nearly the same motion of a vessel can be achievedwith the 0-5-degree motion of the vessel driven by a cam arm on bothsides of the vessel, effectively giving the vessel a +/−5 degrees motionin half the height. Further, the motion of the cam arms 762 (e.g., speedof cam arm rotation and timing between opposing cam arms) can beadjusted to maximize the wave formation in the vessel to maximize waveamplitude and thus (ideally) maximize homogeneity of vessel contents andtime to achieve homogeneity. The timing can also be adjusted based onvolume in a vessel with a given geometry to maximize the mixingefficiency.

In an embodiment, the optical sensor 756 can be used to confirm that thefirst bioreactor vessel 410 has been correctly re-positioned after eachcam agitation motion. It is further contemplated that correctre-positioning of the bioreactor vessel can be checked and verified evenbetween alternating cam motions. This enables quick detection ofmisalignment, in substantially real time, allowing for an operator tointervene to reseat the bioreactor vessel without substantial deviationfrom the bioprocessing operation/protocol.

FIG. 43 is a schematic illustration showing the position of a fluid 800within the bioreactor vessel during this agitating process. As shown inFIG. 42, in an embodiment, a homing sensor 802 integrated with the bedplate 746 may be utilized by the controller to determine when the camarms 762 have returned to the clearance position beneath the top surfaceof the bed pate 746. This is useful in coordinating the motion of thecam arms 762 to provide a desired mixing frequency in the bioreactorvessels. In an embodiment, the cam arms 762 are configured to provide amaximum 5 degree tilting angle with respect to the bed plate 746.

With reference to FIG. 44, the interface between the locating pins 754of the bed plate and the recesses 550 in the bottom plate 502 of thebioreactor vessel 410 during mixing/agitation is illustrated. In anembodiment, the recesses 550 have a dome-like or hemispherical-likeinterior surface and a diameter, d1, that is greater than a diameter,d2, of the locating pins 754. As illustrated in FIG. 44, thisconfiguration provides for clearance between the locating pins 754 andrecesses 550, which allows for tilting of the bioreactor vessel 410 whenthe locating pins 554 are received in the recesses 550.

In an embodiment, each drawer of the bioprocessing apparatus 700, e.g.,drawer 712, desirably includes have a flip-down front panel 810hingedly-mounted thereto, as shown in FIGS. 45-50. The flip-down frontpanel 810 allows access to the auxiliary compartment 730 without havingto open the drawer 712, as best shown in FIGS. 45, 49 and 50. As will beappreciated, this configuration allows for in-process sampling andexchange of media bags. In connection with the above, in an embodiment,the auxiliary compartment 730 may be configured with a plurality oftelescoping sliding rails 812 providing attachment means 815 from whichthe various reservoirs/media bags can be suspended. Rails 812 aremovable between a retracted position within compartment 730, as depictedin FIG. 48, to an extended position out from compartment 730, asdepicted in FIG. 49. When a collection bag is full, or a media/fluid bagneeds replacement, the rails 812 can simply be extended outward and thebag unclipped. A new bag can be connected to its respective tail andthen be suspended from a rail and slid back into the auxiliarycompartment 730 without having to open the drawer 712 or pauseprocessing. In an embodiment, the rails 812 may be mounted ontransversely-extending cross rods 814. The rails 812 may thus belaterally slidable on the rods 814, and extendable from and retractableinto the auxiliary compartment. In addition, the when the drawer is open(FIG. 46) the rails 812 can rotate about the rear cross rod so that itclears the compartment 730 to allow a user to thread the tubing tailstowards the front of the 730 compartment, providing for a third degreeof freedom.

As illustrated in FIG. 51, in another embodiment, the media/fluid bagsmay be mounted on a platform 820 that is rotatable out of the auxiliarycompartment 730 from a stowed position to an access position. Forexample the platform 820 may be mounted for movement along a guide track822 formed in the sidewalls of the auxiliary compartment 730.

With reference to FIG. 52, in an embodiment, the bioprocessing apparatus700 may further include a low-profile waste tray 816 received within thehousing 710 beneath each drawer, e.g., drawer 712. Waste tray 816 isindependently mounted on its drawer to be moveable between a closed andopen position. In the closed position, tray 816 desirably extends flushwith the front surface of the drawer while in the open position tray 816exposes its own chamber 819 to be accessible to an operator. Chamber 819provides for easy storage of large waste bags connected to the fluidpath of its overlying tray 600 and provides access thereto withouthaving to open the drawer 712. In addition, in the closed position, thewaste tray 816 positions chamber 819 in underlying registry with itsdrawer and is sized and shaped so as to be operable to contain any leaksfrom the processing chamber 722 or auxiliary compartment 730.

In an embodiment, each drawer may include a camera positioned aboveprocessing chamber (e.g., above each bioreactor vessel 410, 420) toallow for visual monitoring of the interior of the drawer 712 withouthaving to open the drawer 712. In an embodiment, the camera (or anadditional camera) can be integrated with the bed plate assembly, or ona sidewall looking laterally into the bioreactor vessel(s).

The second module 200 of the invention therefore provides for theautomation of cell processing to an extent heretofore not seen in theart. In particular, the fluid flow architecture 400, pump assembly 738and pinch valve array 736 allows for automated fluid manipulationbetween the bioreactor vessels 410, 420 and the bags connected to thefirst and second fluid assemblies 740, 744 (e.g., fluid addition,transfer, draining, rinsing, etc.). As discussed below, thisconfiguration also permits hollow-fiber filer concentration and wash,filterless perfusion and line priming. The use of the drawer engagementactuator 740 also for automatic engagement and disengagement of thedrop-in kit 600, further minimizing human touchpoints. Indeed, humantouchpoints may only be required for source/media bag addition andremoval, sampling and data input (e.g., sample volume, cell density,etc.).

Referring to FIGS. 53-77, an automated, generic protocol for a workflowwith immobilized Ab coating, soluble Ab addition, gamma-retroviralvector with expansion in the same vessel, using the second module 200and fluid flow architecture 400 thereof, is illustrated. This genericprotocol provides for activation (illustrated in FIG. 53-59),pre-transduction preparation and transduction (illustrated in FIGS.60-71), expansion (FIGS. 72-76), and, for some embodiments, harvesting(FIG. 77) of a population of cells in an automated andfunctionally-closed manner. In describing operation of the pinch valves,below, when a valve is not used for a particular operation, the valve isin its closed state/position. Accordingly, after a valve is opened toallow for a particular operation, and once that operation is completed,the valve is closed before proceeding to the next operation/step.

As shown in FIG. 53, in a first step, valves 432 and 468 f are openedand first fluid assembly line pump 454 is actuated to pump an antibody(Ab) coating solution from reservoir 466 f connected to the first fluidassembly 440 to the first bioreactor vessel 410 through the first port412 thereof. The antibody coating solution is incubated for a period oftime, and then drained through the interconnect line to a wastereservoir 472 a of the first fluid assembly 440 by opening valves 434,474 a and activating the circulation line pump 456. As described herein,draining of the bioreactor vessel 410 may be facilitated by tilting thebioreactor vessel 410 using the cam arms 462.

After draining the antibody coating solution, valves 432 and 468 e areopened and pump 454 is actuated to pump a rinse buffer from reservoir466 e connected to the first fluid assembly 440 to the first bioreactorvessel 410 through the first bioreactor line. The rinse buffer is thendrained through interconnect line 450 to the waste reservoir 472 a byactuating the circulation line pump 456 and opening valve 474 a. In anembodiment, this rinse and draining procedure may be repeated multipletimes to adequately rinse the first bioreactor vessel 410.

Turning to FIG. 55, after rinsing the first bioreactor vessel 410 withthe buffer, cells in a seed bag 466 d (which have been previouslyenriched and isolated using the first module 100) are transferred to thefirst bioreactor vessel by opening valves 468 d and 432, and actuatingthe pump 454. The cells are pumped through the first bioreactor line 414of the first bioreactor vessel 410 and enter the bioreactor vessel 410through first port 412. As shown in FIG. 56, valves 432 and 468 a arethen opened and pump 454 is actuated to pump a second antibody (Ab)solution from reservoir 466 a connected to the first fluid assembly 440to the first bioreactor vessel 410 through the first port 412.

After pumping the second antibody solution into the first bioreactorvessel, the second antibody solution reservoir 466 a is then rinsed andthe rinse media is pumped to the first bioreactor vessel. In particular,as shown in FIG. 57, valves 474 b, 452 and 468 a are opened and rinsemedia from a rinse media reservoir/bag 472 b of the second fluidassembly 444 is pumped using pump 454 into the second antibody solutionreservoir 466 a to rinse the reservoir. After rinsing, valve 432 isopened and the rinse media is pumped from the reservoir 466 a to thefirst bioreactor vessel 410. In an embodiment, second antibody solutionreservoir 466 a may be rinsed multiple times using this procedure.

After rinsing the second antibody solution reservoir 466 a, theinoculum/seed cell bag 466 d may also be optionally rinsed. Inparticular, as shown in FIG. 58, valves 474 b, 452 and 468 d are openedand rinse media from a rinse media reservoir/bag 472 b of the secondfluid assembly 444 is pumped into the inoculum/seed cell bag 466 d torinse the bag using pump 454. After rinsing, valve 432 is opened and therinse media is pumped from the bag 466 d to the first bioreactor vessel410 using pump 454. By pumping the rinse media to the first bioreactorvessel 410 after rinsing the inoculum/seed cell bag 466 d, the celldensity in the first bioreactor vessel 410 is reduced. At this time, asample may be taken to measure one or more parameters of the solution inthe first bioreactor vessel prior to activation (e.g., to ensure adesired cell density is present prior to activation. In particular, asshown in FIG. 58, valves 434, 452 and 432 are opened and pump 456 isactuated to pump the contents of the first bioreactor vessel 410 along afirst circulation loop of the first bioreactor vessel (i.e., out of thesecond port 416, through the interconnect line 450, and back to thefirst bioreactor vessel 410 through the first bioreactor line 414 andfirst port 412 of the first bioreactor vessel 410). To take a sample, afirst sample vessel 280 a (e.g., a dip tube, syringe, etc.) is connectedto the first sample tubing tail 476 a and valve 478 a is opened todivert some of the flow through the interconnect line 450 to the firstsample vessel 280 a for analysis.

If analysis of the sample taken indicates that all solution parametersare within predetermined ranges, then the solution within the firstbioreactor vessel 410 is incubated for a predetermined period of timefor activation of the population of cells in solution, as illustrated inFIG. 59. For example, in an embodiment, the population of cells in thefirst bioreactor vessel 410 may be incubated for approximately 24-48hours.

Referring now to FIG. 60, after activation, to prepare for transduction,valves 438 and 474 b may be opened and pump 456 operated to pump theRetroNectin solution from reservoir 472 b to the second bioreactorvessel 420 through the second port 426 of the second bioreactor vessel420. After pumping the RetroNectin solution to the second bioreactorvessel 420 for RetroNectin coating of the second bioreactor vessel 420,the solution is incubated in the second bioreactor vessel 420 for apredetermined time period. As further shown in FIG. 60, afterincubation, all RetroNectin solution is then drained from the secondbioreactor vessel 420 to the waste reservoir 472 a by opening valves 438and 474 a and actuating the circulation line pump 456. During theseRetroNectin coating, incubation and draining steps (relating to thesecond bioreactor vessel 420), it should be noted that the activatedcell population remains in the first bioreactor vessel 410. It should benoted that it is not necessary that RetroNectin or other reagents forenhancing the efficiency of genetic modification be utilized in allprocesses.

As shown in FIG. 61, after RetroNectin coating, a rinse buffer bag 472 bis connected to the second fluid assembly 444 (or it may already bepresent and connected to one of the tubing tails), and valves 474 b and438 are opened and pump 456 is actuated to pump buffer from the bag 472b to the second bioreactor vessel 420. As discussed above,alternatively, the buffer may be pumped through the first port 422 ofthe second bioreactor vessel 420 by instead opening valves 452 and 436.

Turning now to FIG. 62 after a defined period of time, all buffer in thesecond bioreactor vessel 420, is drained to the waste reservoir 472 a ofthe second fluid assembly 444 by opening valves 438 and 474 a andactuating the interconnect line pump 456.

At this point, as shown in FIG. 63, a post-activation pre-concentrationsample may be taken of the cells in the first bioreactor vessel 410. Asshown therein, valves 434, 486, 488 and 432 are opened and pump 456actuated to circulate the solution in the first bioreactor vessel 410out of the second port 434, through the interconnect line, through thefiltration line 48 and filter 484, through the first bioreactor line 414of the first bioreactor vessel 410, and back to the first bioreactorvessel 410 through the first port 412. To take a sample, a second samplevessel 280 b (e.g., a dip tube, syringe, etc.) is connected to thesecond sample tubing tail 476 b and valve 478 b is opened to divert someof the flow through the interconnect line 450 to the second samplevessel 280 b for analysis.

Referring now to FIG. 64, and depending on the concentration obtainedfrom the sample, concentration may be carried out by circulating thecontents of the first bioreactor vessel 410 trough the filter 484. Asdiscussed above, this is accomplished by opening valves 434, 486, 488and 432 and actuating pump 456, which causes circulation of the solutionin the first bioreactor vessel 410 out of the second port 416, throughthe second bioreactor line 418, through the interconnect line 450,through the filtration line 482 and filter 484, through the firstbioreactor line 414 of the first bioreactor vessel 410, and back to thefirst bioreactor vessel 410 through the first port 412. As the fluidpasses through the filter 484, waste is removed and permeate pump 492pumps such waste to the waste reservoir 472 a of the second fluidassembly 444 through waste line 490. In an embodiment, this procedure isrepeated until the volume in the first bioreactor vessel 410 isconcentrated to a predetermined volume.

Turning to FIG. 65 after concentration, the concentrated cell populationin the activation vessel (i.e., first vessel 410 containing aconcentrated cell population) is washed at constant volume throughperfusion. In particular, as shown therein, media from a media bag 466 bof the first fluid assembly 440 is pumped into the first bioreactorvessel 410 through first port 412 through the interconnect line 450 atthe same time as media is pumped out of the first bioreactor vessel 410though the second port 416 such that a constant volume is maintained inthe first bioreactor vessel 410. As the media is added and removed fromthe vessel 410, waste may be filtered out by filter 484 and directed tothe waste reservoir 472 a.

A post-wash sample may be taken of the cells in the first bioreactorvessel 410 in a manner similar to that previously described forpre-concentration sampling. In particular, as shown in FIG. 66, valves434, 486, 488 and 432 are opened and pump 456 actuated to circulate thefluid in the first bioreactor vessel 410 out of the second port 434,through the interconnect line, through the filtration line 48 and filter484, through the first bioreactor line 414 of the first bioreactorvessel 410, and back to the first bioreactor vessel 410 through thefirst port 412. To take a sample, a third sample vessel 280 c (e.g., adip tube, syringe, etc.) is connected to the third sample tubing tail476 c and valve 478 c is opened to divert some of the flow through theinterconnect line 450 to the third sample vessel 280 c for analysis.

As shown in FIG. 67, a bag containing a thawed viral vector is connectedto the first fluid assembly 440, such as through tubing tail 464 c.Valves 468 c and 436 are then opened and pump 454 actuated to transferthe viral vector coating solution from the bag 466 c to the secondbioreactor vessel 420 through first port 422. Incubation is then carriedout for a predetermined period of time, for virus coating of the secondbioreactor vessel 420. Subsequent to incubation, the viral vectorcoating solution is drained from the second bioreactor vessel 420 to thewaste reservoir 472 a by opening valves 438 and 474 a and actuating thecirculation line pump 456. In embodiments, viral and non-viral vectorscan be utilized as agents for transduction/genetic modification.

As illustrated in FIG. 68, after the second bioreactor vessel 420 iscoated with the viral vector, the post-wash cells from the firstbioreactor vessel 410 are transferred to the second bioreactor vessel420 for transduction/genetic modification. In particular, valves 434,452 and 436 are opened and the circulation line pump 456 is actuated topump the cells out of the first bioreactor vessel 420 through the secondport 416 of the first bioreactor vessel 410, through interconnect line450, to the first bioreactor line 424 of the second bioreactor vessel420, and into the second bioreactor vessel 420 through the first port422 of the second bioreactor vessel 420.

Media from media bag 466 b is then added to the second bioreactor vessel420 by opening valves 468 b and 436 and actuating pump 454 to increasethe total volume of the solution in the second bioreactor vessel 420 toa predetermined volume, as illustrated in FIG. 69. With reference toFIG. 70, a pre-transduction sample may then be taken by opening valves438, 452 and 436 and actuating the circulation line pump 456 to pump thesolution in the second bioreactor vessel 420 along a circulation loop ofthe second bioreactor vessel (i.e., out of the second port 426, throughthe interconnect line 450, and back to the second bioreactor vessel 420through the first bioreactor line 414 and first port 422 of the secondbioreactor vessel 420). To take a sample, a fourth sample vessel 280 d(e.g., a dip tube, syringe, etc.) is connected to the fourth sampletubing tail 476 d and valve 478 d is opened to divert some of the flowthrough the interconnect line 450 to the fourth sample vessel 280 d foranalysis.

If analysis of the fourth sample taken indicates that all parameters arewithin predetermined ranges required for successful transduction, thenthe population of cells within the second bioreactor vessel 420 isincubated for a predetermined period of time for transduction of thepopulation of cells in solution, as illustrated in FIG. 71. For example,in an embodiment, the population of cells in the second bioreactorvessel 420 may be incubated for 24 hours for transduction.

With reference to FIG. 72, after transduction, media is added to thesecond bioreactor vessel 420 to achieve a predetermined expansion volumein the second bioreactor vessel 420. As shown therein, to add media,valves 468 b and 436 are opened and pump 454 is actuated to pumpgrowth/perfusion media from media bag 466 b to the second bioreactorvessel 420 through the first port 422 of the second bioreactor vesseluntil the predetermined expansion volume is reached.

As illustrated in FIG. 73, a pre-expansion sample may then be taken byopening valves 438, 452 and 436 and actuating the circulation line pump456 to pump the solution in the second bioreactor vessel 420 along thecirculation loop of the second bioreactor vessel 420, as indicated above(i.e., out of the second port 426, through the interconnect line 450,and back to the second bioreactor vessel 420 through the firstbioreactor line 414 and first port 422 of the second bioreactor vessel420). To take a sample, a fifth sample vessel 280 e (e.g., a dip tube,syringe, etc.) is connected to the fifth sample tubing tail 476 e andvalve 478 e is opened to divert some of the flow through theinterconnect line 450 to the fifth sample vessel 280 e for analysis.

If analysis of the fifth sample taken indicates that all parameters arewithin predetermined ranges required for successful expansion of thepopulation of cells, then the population of cells within the secondbioreactor vessel 420 is incubated for a predetermined period of time,e.g., 4 hours, to let the cells settle.

Subsequent to this incubation period or at a later predetermined time,perfusion at a rate of 1 volume per day (1× perfusion) is carried out bypumping media from media bag 466 b into the second bioreactor vessel 420through first port 422 at the same time as spent/used media is pumpedout of the second bioreactor vessel 420 though the second port 426 (andthrough interconnect line 450 to the waste reservoir 472 a), as shown inFIG. 74. This perfusion is accomplished by opening valves 468 b, 436,438 and 474 a, and actuating the first pump 454 and circulation linepump 456. During this 1× perfusion, the media from media bag 466 b isintroduced into the second bioreactor vessel 420 at substantially thesame rate as used media is removed from the second bioreactor vessel 420and sent to waste, to maintain a substantially constant volume withinthe second bioreactor vessel 420.

Sampling may then be carried out as needed/desired to monitor theexpansion process and/or to determine when a desired cell density isreached. As discussed above, samples may be taken by opening valves 438,452 and 436 and actuating the circulation line pump 456 to pump thesolution in the second bioreactor vessel 420 along the circulation loopof the second bioreactor vessel 420, as indicated above (i.e., out ofthe second port 426, through the second bioreactor line 428, through theinterconnect line 450, and back to the second bioreactor vessel 420through the first bioreactor line 424 and first port 422 of the secondbioreactor vessel 420). To take a sample, another sample vessel 280 x(e.g., a dip tube, syringe, etc.) is connected to a sample tubing tailof the sample assembly 448 and a valve of the tubing tail is opened todivert some of the flow through the interconnect line 450 to the samplevessel 280 x for analysis, as shown in FIG. 75. After each samplingoperation, incubation without perfusion is carried out for apredetermined time period, e.g., four hours, to allow the cells tosettle before restarting perfusion.

As shown in FIG. 76, Subsequent to this incubation period, perfusion ata rate of 1 volume per day (1× perfusion) is carried out by pumpingmedia from media bag 466 b into the second bioreactor vessel 420 throughfirst port 422 at the same time as spent/used media is pumped out of thesecond bioreactor vessel 420 though the second port 426 (and throughinterconnect line 450 to the waste reservoir 472 a), as shown in FIG.74. This perfusion is accomplished by opening valves 468 b, 436, 438 and474 a, and actuating the first pump 454 and circulation line pump 456.

When sampling indicates a viable cell density (VCD) of a predeterminedthreshold value (e.g., 5 MM/mL), perfusion at a rate of 2 volumes perday (2× perfusion) is carried out by pumping media from media bag 466 binto the second bioreactor vessel 420 through first port 422 at the sametime as spent/used media is pumped out of the second bioreactor vessel420 though the second port 426 (and through interconnect line 450 to thewaste reservoir 472 a), as shown in FIG. 76. This perfusion isaccomplished by opening valves 468 b, 436, 438 and 474 a, and actuatingthe first pump 454 and circulation line pump 456. During this 2×perfusion, the media from media bag 466 b is introduced into the secondbioreactor vessel 420 at substantially the same rate as used media isremoved from the second bioreactor vessel 420 and sent to waste, tomaintain a substantially constant volume within the second bioreactorvessel 420.

Finally, with reference to FIG. 77, after a desired, viable cell densityis achieved, the cells may be harvested by opening valves 438 and 474 dand actuating the circulation line pump 456. The expanded population ofcells is then pumped out of the second bioreactor vessel 420 through thesecond port 426, through interconnect line 450, and to a collection bag472 d connected to the tubing tail 470 d of the second tubing assembly444. These cells can then be formulated in a manner heretofore known inthe art for delivery and infusion into a patient.

The second module 200 of the bioprocessing system 10, and the flowarchitecture 400 and bioreactor vessels 410, 420 thereof, thereforeprovides for a flexible platform on which a variety of bioprocessingoperations may be carried out in a substantially automated andfunctionally closed manner. In particular, while FIGS. 53-77 illustratean exemplary generic protocol that can be carried out using thebioprocessing system 10 of the invention (particularly, using the secondmodule 200 thereof), the system is not so limited in this regard.Indeed, various automated protocols can be enabled by the system of theinvention, including a number of customer-specific protocols.

In contrast to existing systems, the second module 200 of thebioprocessing system 10 is a functionally-closed, automated system thathouses the first and second bioreactor vessel 410, 420 and the fluidhandling and fluid containment systems, which are all maintained atcell-culture friendly environmental conditions (i.e., within atemperature and gas-controlled environment) to enable cell activation,transduction and expansion. As discussed above, the system includesautomated kit loading and closed sampling capability. In thisconfiguration, the system enables all steps of immune cell activation,transduction, expansion, sampling, perfusion and washing in a singlesystem. It also provides the user the flexibility of combining all stepsin a single bioreactor vessel (e.g., first bioreactor vessel 410) orusing both of the bioreactor vessels 410, 420 for end-to-end activationand washing. In an embodiment, a single expansion bioreactor vessel(e.g., bioreactor vessel 420) is capable of robustly generating a doseof billions of T cells. Either single or multiple doses can be generatedin situ with high recovery and high viability. In addition, the systemis designed to give the end-user the flexibility of running differentprotocols for the manufacture of genetically modified immune cells.

Some of the commercial advantages provided by the bioprocessing systemof the invention include robust and scalable manufacturing technologyfor product commercialization by simplifying workflows, reducing laborintensity, reducing the burden on clean room infrastructure, reducingfailure nodes, reducing costs and the ability to increase scale ofoperations.

As discussed above in connection with the generic workflow, the systemof the invention, the bioprocessing system 10, and the flow architecture400 and bioreactor vessels 410, 420 of the second module 200 provide forculture concentration, washing, slow perfusion, fast perfusion, and‘round robin’ perfusion processes to be carried out in an automated andfunctionally-closed manner. For example, as discussed above, the pump456 on the interconnect line 450 can be used to circulate the fluid fromone of the ports of the bioreactor through the filtration line 482 andfilter 484 and then back to another port on the bioreactor, whilerunning the permeate pump 492 (typically at a percentage of thecirculation pump 456, such as for example, about 10%], in aconcentration step. The concentration can be run open loop, or can bestopped based on a measured volume removed from the bioreactor or ameasured volume accumulated in the waste. In an embodiment, the filter,pump speeds, filter area, number of lumens, etc. are all sizedappropriately for total number of cells and target cell density to limitfouling and excessive cell loss due to shear.

In an embodiment, and as discussed above, the system of the inventioncan also be used for washing, e.g., to remove residuals such asremaining viral vector after incubation. Washing involves the same stepsdescribed above for concentration, except the pump 454 on the firstfluid assembly line 442 is used to pump in additional culture media toreplace the fluid pumped from the permeate waste pump 492. The rate ofintroduction of new medium can correspond to the rate of removal offluid by the permeate pump 492. This allows for a constant volume to bemaintained in the bioreactor vessel, and residuals can be removedexponentially with time so long as the contents in the bioreactor arewell mixed (circulation may suffice). In embodiments, this same processcan be utilized post activation for the in-situ hollow fiberfiltration-based washing of the cell suspension to remove residuals. Forcoated and non-coated surfaces, the soluble activation reagent washoutcan also be done via filter-based perfusion.

As also discussed above, the pump 454 on the first fluid assembly line442 can be used to add media to a given bioreactor vessel while the pump456 on the interconnect line 450 is used to move spent media to thewaste bag in the second fluid assembly, in a perfusion process. In anembodiment, gravity can be used to settle the cells, and the spent mediacan be pumped out at such a rate so as not to significantly disturb thecells within the bioreactor vessel. This process may involve running thepumps 454 and 456 open loop at the same rate. In an embodiment, one pump(454 or 456) may be run at a set rate, and the rate of the other pumpmay be adjusted based on the mass/volume of the bioreactor vessel or themass/volume of the waste bag (or the mass/volume of a measured sourcebag).

In connection with the above, it is contemplated that pump control maybe based on a weight measurement of the bioreactor vessels (using thefeedback from the load cells 760). For example, the configuration of thesystem enables on-the-fly pump calibration based on load cell readings,allowing the system to automatically accommodate changes in thetube/pump performance over time. Further, this method can be used forclosed loop control on a mass (volume) rate of change when emptying orfilling a bioreactor vessel.

FIG. 81 illustrates one exemplary embodiment of a method 480 ofutilizing the second module 200 in a perfusion process. The method 480includes activating a first pump 454 to pump fresh media to thebioreactor vessel 410 containing a genetically modified population ofcells, at 482, activating a second pump 456 to pump spent media from thebioreactor vessel 410 to a waste bag 472 a, at 484, acquiring mass datarelating to a mass of the bioreactor vessel (e.g., bioreactor vessel410) using the load cells associated with the bed plate, at 486,determining whether or not the mass of bioreactor vessel 410 has changedor remains substantially constant, at 488, and if the mass of thebioreactor vessel has changed, adjusting an operational parameter of atleast one of the first pump and the second pump to maintain asubstantially constant mass of the bioreactor vessel 410, at 490. Forexample, if it is determined that the mass of the bioreactor vessel 410has decreased, this indicates that the spent media is being removed fromthe bioreactor vessel at a rate greater than the rate of addition offresh media to the bioreactor vessel. Accordingly, and in response, theflow rate of the first pump may be increased and/or the flow rate of thesecond pump may be decreased to maintain a substantially constant mass(and volume) in the bioreactor vessel 410. Further mass data may then beacquired and further adjustments to pump operation made, if necessary,to maintain a substantially constant mass/volume in the bioreactorvessel 410. If the mass is determined to be substantially constant aftersome period of time of operation of the first and second pumps, thepumps may be maintained at their current operational setpoints (e.g.,flow rates), as shown at 492.

In another embodiment, the bioprocessing system allows for round-robinperfusion of the various bioreactor vessels in the system using the flowarchitecture 400. For example, the circulation pump 456 and the pump 545along the first fluid assembly line 442 are used to perfuse cells withinthe first bioreactor vessel 410 in conjunction with the appropriatepinch valve states, as described above. Perfusion of the cells withinthe first bioreactor vessel 410 may then be ceased or paused, and thenthe circulation pump 456 and the pump 454 and appropriate pinch valvesmay be actuated to perfuse cells within the second bioreactor vessel420. In this respect, perfusion of the various bioreactors can beperformed sequentially (i.e., perfusion of the first bioreactor vessel410 for a period of time, then perfusion of the second bioreactor vessel420 for period of time, in a repeating and alternating manner). Thisallows for perfusion of any number of bioreactor vessels in the systemwithout requiring the use of more pumps, media bags or waste bags.

With round-robin perfusion, the pumps could run continuously, could berun intermittently together (duty cycle), or could be run sequentially(source, then waste, repeat), so as still maintain the volume/mass inthe various bioreactor vessels at about the same level. Round robinperfusion (intermittently running the set of pumps together and waitingan interval of time) would also allow for perfusion of multiple vesselsusing the same two pumps, as indicated. Further, round robin perfusionallows for a lower effective exchange rate (such as about 1 Vol/day)even if the pumps don't have a great low-end dynamic range. Further,round-robin perfusion also allows each vessel to be perfused withdifferent medium as controlled by the valves in the first fluid assembly440.

In addition, in an embodiment, fast perfusion can be used for residualremoval (e.g., for post activation Ab removal and/or post transductionresidual removal). In a fast-perfusion process, the perfusion processdescribed above may be run much faster than the typical 1-5 volumes/day,such as, for example, between about 8-20 volumes/day, or greater thanabout 20 volumes/day to achieve 1 log reduction in a matter of minutesto hours. In an embodiment, the perfusion rate is balanced against cellloss. In some embodiment, fast perfusion may allow for the eliminationof the hollow filter 484 and still meet biological imperatives ofquickly removing residuals after certain steps.

As further described above, the system of the invention facilitatesrinsing a bag/reservoir connected to the first fluid assembly 440 usinga rinse buffer or fluid from another bag/reservoir connected to thesecond fluid assembly 444 using the pump 454 on the first fluid assemblyline 442. In addition, the fluid lines of the flow architecture/system400 can be cleared with sterile air from the sterile air source 458 toprevent cells from sitting in the lines and dying or to prevent mediumor reagents from sitting in the lines and degrade or go unused. Thesterile air source 458 can also be used to clear out reagents from thelines so as to ensure that no more reagent is pumped to the bioreactorvessels 410, 420 than intended. The sterile air source 458 can likewisebe used to clear lines all the way to the connected bag (of the first orsecond fluid assembly 440, 444) to clear for sterile tube welding tolimit carryover. Alternatively, or in addition to clearing lines usingthe sterile air source 458, lines may be cleared using air pulled fromone of the bioreactor vessels so long as the port through which the airis pulled is not immersed and the bioreactor vessel has an air balanceport 530.

As discussed above, the system allows for closed-drawer, in processsampling of the contents of the bioreactor vessel(s). During sampling,the vessel from which the sample is to be pulled may be agitated usingthe cam arms 762, circulating the contents of the vessel using thecirculation line pump 456, and using the sampling assembly 448 towithdraw a sample from the interconnect line 450. In an embodiment, onlynon-bead bound cells may be agitated.

As also discussed above, the system of the invention allows for thepopulation of cells to be collected after a target cell density isachieved. In an embodiment, collecting the expanded population oftransduced cells may include moving cells to one of the bags connectedto the second fluid assembly 444 using the pump 456 on the interconnectline 450, or circulating the cells with interconnect pump 456 to movethe cells to a bag connected to the first fluid assembly 440. Thisprocess could be used for final collection or for a large sample volume,or could be used to fully automate the sampling process (i.e., byconnecting a syringe or bag to the first fluid assembly 440, circulatingcontents of the bioreactor vessel, and pulling in a portion of a desiredsample volume from the circulated contents with fluid assembly pump 454and moving towards syringe/bag). In such a case, the circulation pump456 and valves can then be used to clear circulation lines offluid/cells. In addition, the pump 454 on the first fluid assembly line442 can be used to continue to push all of the aliquoted sample volumeto the sample container, using the air in the line to complete to sampletransfer to the container without an appreciable amount of cellsremaining in the lines.

While the embodiments described above disclose a workflow whereactivation of cells is carried out in a first bioreactor vessel and theactivated cells are transferred to the second bioreactor vessel fortransduction and expansion, in an embodiment, the system of theinvention may allow for activation and transduction operations to becarried out in a first bioreactor vessel, and expansion of thegenetically modified cells carried out in a second bioreactor vessel.Moreover, in an embodiment, the system of the invention may allow forthe in-situ processing of isolated T cells wherein the activation,transduction and expansion unit operations are all performed within asingle bioreactor vessel. In an embodiment, the invention thereforsimplifies existing protocol by enabling a simplified andautomation-friendly ‘one-pot’ activation, transduction and expansionvessel.

In such an embodiment, the T-cell activator may be micron-sizedDynabeads and a lentiviral vector is used for transduction. Inparticular, as disclosed therein, micron-sized Dynabeads serve the dualpurpose of isolating and activating T cells. In an embodiment,activation (and isolation) of the T cells may be carried out in one ofthe bioreactor vessels 410 using Dynabeads in the manner indicatedabove. Subsequently, the activated cells are transduced by viruses forgenetic modification, such as in the manner described above inconnection with FIGS. 60-71. Post-activation and viral transduction, thevirus may then be washed out of the bioreactor vessel 410 using thefilterless perfusion method described above that retains the cells andthe micron-sized Dynabeads in the bioreactor vessel 410. This enablescell expansion in the same bioreactor vessel 410 that is used foractivation and transduction. The filterless perfusion methodadditionally enables the culture wash to take place without the need forfirst immobilizing the activation beads that need to be retained alongwith the cells during expansion. In particular, when the virus is washedout, the micron size Dynabeads are not fluidized in the slow perfusionrate and are retained in the vessel. Nanometer sized viral particles andresidual macromolecules are fluidized during the slow perfusion and arewashed out.

In an embodiment, after expansion, the cells may be harvested in themanner described above in connection with FIG. 77. After harvest, amagnetic debeading process may be utilized to remove the Dynabeads fromthe collected cells. In other embodiments, the steps of harvesting theexpanded population of cells and debeading the cells are carried outsimultaneously using perfusion, whereby culture media is introducedthrough a feed port in the bioreactor vessel while cell culture mediumincluding the expanded population of cells is removed from thebioreactor vessel through a drain port in the bioreactor vessel. Inparticular, when final debeading of the culture is required, filterlessperfusion can be used to debead the micron-sized beads by takingadvantage of the difference in weight of the cells and that of thecell-Dynabead complexes. In order to debead the culture, the entirecontents of the bioreactor vessel would be mixed (using, for examplingthe cam arms 762 of the actuator mechanism in the manner hereinbeforedescribed). After mixing/agitation, the heavy Dynabeads would sink andsettle on the silicon membrane 516 within 10-15 minutes. In contrast,the cells need over 4 hours to settle down over the membrane 516. Aftera hold period of 10-15 minutes post mixing/agitation, the cellsuspension can be slowly pulled out using perfusion without disturbingthe settled Dynabeads. The incoming medium line may be used to maintainthe medium bed height within the bioreactor vessel. Thus inventiondescribed herein simplifies the current Dynabeads protocol byeliminating the need for several mid-process cell transfers and discreetwashing and debeading steps, and minimizes costs and potential risks. Bydebeading the culture at the same time as harvesting the cells, the needof additional magnetic devices or disposables, which have typically beennecessary, can be eliminated.

In contrast to other static, perfusion-free culture systems, thegas-permeable membrane-based bioreactor vessel 410 of the inventionsupports high density cell culture (e.g., up to 35 mm/cm²). Thus, allfour unit processes of activation using Dynabeads, transduction, washingand expansion can be performed in the same bioreactor vessel, in a fullyautomated and functionally-closed manner. The bioprocessing system ofthe invention therefore simplifies current protocol by eliminating theneed for mid-process cell transfer and discreet washing steps, andminimizes costs and potential risks resulting from multiple humantouchpoints.

In an embodiment, the two bioreactor vessels 410, 420 of the system canbe run with either the same starting culture or two simultaneous splitcultures, e.g., CD4+ cells in one bioreactor vessel 410, and CD8+ cellsin the other bioreactor vessel 420. A split culture allows the parallelindependent processing and expansion of two cell types that can becombined prior to infusion into the patient.

While a number of possible CAR-T workflows for the generation andexpansion of genetically modified cells using the bioprocessing systemof the invention have been described above, the workflows describedherein are not intended to be comprehensive, as other CAR-T workflowsare also enabled by the system of the invention. In addition, while thesystem of the invention and, in particular, the second module 200 of thesystem, has been described in connection with the manufacture of CAR-Tcells, the system of the invention is also is compatible with themanufacture of other immune cells, such TCR-T cells and NK cells.Moreover, while embodiments of the invention, disclose the use of thetwo bioreactor vessels 410, 420 in a two-step, sequential process wherethe output of the first bioreactor vessel 410 is added to the secondbioreactor vessel 420 for additional processing steps (e.g., activationin the first bioreactor vessel and transduction and expansion in thesecond bioreactor vessel), in some embodiments, the two bioreactorvessels can be used for identical workflows in duplicate. Examplereasons for using a second bioreactor vessel sequentially can includeresidual chemical modifications (e.g., coatings or immobilized reagents)that cannot be washed out of the first bioreactor that are detrimentalin later steps or if overexposure of cells occurs in earlier steps, or aneed to pre-coat a bioreactor surface prior to the addition of cells(e.g., RetroNectin coating).

Additional examples of potential single bioreactor vessel workflows thatare enabled by the system of the invention include (1) soluble activatoractivation, viral transduction, filterless perfusion and expansion in asingle bioreactor vessel, (2) Dynabead-based activation, viraltransduction, filterless perfusion and expansion in a single bioreactorvessel and (3) TransAct bead-based activation, viral transduction,filterless perfusion and expansion in a single vessel.

Moreover, further examples of potential multiple bioreactor vesselworkflows that are enabled by the system of the invention include (1)soluble activator activation, viral transduction, filterless perfusionand expansion in the first bioreactor vessel 410, and soluble activatoractivation, Lentiviral transduction, filterless perfusion and expansionin the second bioreactor vessel 420, using identical cell types or splitcultures in the two bioreactor vessels; (2) Dynabead-based activation,viral transduction, filterless perfusion and expansion in the firstbioreactor vessel 410, and Dynabead-based activation, Lentiviraltransduction, filterless perfusion and expansion in the secondbioreactor vessel 420, using identical cell types or split cultures inthe two bioreactor vessels; (3) TransAct bead-based activation, viraltransduction, filterless perfusion and expansion in the first bioreactorvessel 410, and TransAct-based activation, Lentiviral transduction,filterless perfusion and expansion in the second bioreactor vessel 420,using identical cell types or split cultures in the two bioreactorvessels; (4) soluble activator activation in the first bioreactor vessel410, and RetroNectin coating, transduction and expansion in the secondbioreactor vessel 420; (5) immobilized activator activation in the firstbioreactor vessel 410, and RetroNectin coating, transduction andexpansion in the second bioreactor vessel 420; (6) Dynabead activationin the first bioreactor vessel 410, and RetroNectin coating,transduction and expansion in the second bioreactor vessel 420; (7)Dynabead activation and Lentiviral transduction in the first bioreactorvessel 410, and expansion in the second bioreactor vessel 420; (8)TransAct activation in the first bioreactor vessel 410, and RetroNectincoating, transduction and expansion in the second bioreactor vessel 420;(9) soluble activator activation in the first bioreactor vessel 410, andexpansion of ex-situ electroporated cells or other non-viral modifiedcells in the second bioreactor vessel 420; (10) TransAct activation inthe first bioreactor vessel 410, and expansion of ex-situ electroporatedcells or other non-viral modified cells in the second bioreactor vessel420; (11) Dynabead activation in the first bioreactor vessel 410, andexpansion of ex-situ electroporated cells or other non-viral modifiedcells in the second bioreactor vessel 420; (12) expansion of allogenicNK cells in the first bioreactor vessel 410, and expansion of allogenicNK cells in the second bioreactor vessel 420 (small molecule-basedexpansion, with no genetic modification; (13) expansion of allogenic NKcells in the first bioreactor vessel 410, and expansion of allogenic NKcells in the second bioreactor vessel 420 (feeder cell-based expansion,with no genetic modification); and (14) soluble activator activation,viral transduction, filterless perfusion and expansion of allogenicCAR-NK or CAR-NK 92 cells in the first bioreactor vessel 410 and/or thefirst and second bioreactor vessels 410,420 (with no RetroNectincoating, and where Polybrene is used to assist in transduction).

While the embodiments described above illustrate process monitoringsensors that are integrated with the bioreactor vessels and/or the bedplate (e.g., on the membrane, integrated in the membrane, on the vesselsidewall, etc.), in other embodiments it is contemplated that additionalsensor may be added to the fluid architecture 400, e.g., along the fluidflow lines themselves). These sensors may be disposable-compatiblesensors for monitoring parameters such as pH, dissolved oxygen,density/turbidity (optical sensor) conductivity and viability within thecirculated fluids. By arranging the sensors in the circulation loop(e.g., the circulation loop of the first bioreactor vessel and/or thecirculation loop of the second bioreactor vessel), the vesselconstruction can be simplified. Additionally, in some embodiments, thesensors along the circulation loop may provide more accuraterepresentation of vessel contents when circulated (rather than measuringwhen the cells are static within the vessel). Still further, a flow ratesensor (e.g., ultrasound based) may be added to the flow loop to measurepumping performance and used in conjunction with an algorithm to correctpumping parameters, as necessary.

As indicated above, the first and third modules 100, 300 may take anyform of any system or device(s) known in the art that is capable of cellenrichment and isolation, and harvesting and/or formulation. FIG. 78illustrates one possible configuration of a device/apparatus 900 whichmay be used in the bioprocessing system 10 as the first module 100, forcell enrichment and isolation using various magnetic isolation beadtypes (including, for example, Miltenyi beads, Dynabeads and StemCellEasySep beads). As shown therein, the apparatus 900 includes a base 910that houses a centrifugal processing chamber 912, a high dynamic rangeperistaltic pump assembly 914, a small internal diameter pump tube 916received by the peristaltic pump assembly, a stopcock manifold 918,optical sensors 920, and a heating-cooling-mixing chamber 922. Asindicated below, the stopcock manifold 918 provides a simple andreliable means of interfacing multiple fluid or gas lines togetherusing, for example, luer fittings. In an embodiment, the pump 914 israted to provide flow rates as low as about 3 mL/min and as high asabout 150 mL/min).

As further shown in FIG. 78, the apparatus 900 may include a generallyT-shaped hanger assembly 924 that extends from the base 910 and includesa plurality of hooks 926 for suspending a plurality of processing and/orsource vessels or bags. In an embodiment, there may be six hooks. Eachhook may include an integrated weight sensor for detecting a weight ofeach vessel/bag. In an embodiment, the bags may include a sample sourcebag 930, a process bag 932, an isolation buffer bag 934, a washing bag936, a first storage bag 938, a second storage bag 940, a post-isolationwaste bag 942, a washing waste bag 944, a media bag 946, a release bag948 and a collection bag 950.

The apparatus 900 is configured to be used with, or include, a magneticcell isolation holder 960, as provided herein. The magnetic cellisolation holder 960 may be removable coupled to a magnetic fieldgenerator 962 (e.g., magnetic field plates 964, 966). The magnetic cellisolation holder 960 accommodates a magnetic retention element ormaterial 968, such as a separation column, matrix or tube. In anembodiment, the magnetic cell isolation holder 960 may be constructed asdisclosed in U.S. patent application Ser. No. 15/829,615, filed on Dec.1, 2017, which is hereby incorporated by reference herein in itsentirety. The apparatus 900 may be under control of a controller (e.g.,controller 110), operating according to instructions executed by aprocessor and stored in memory. Such instructions may include themagnetic field parameters. In an embodiment, the apparatus 900 mayfurther include a syringe 952 that can be utilized for bead addition, asdiscussed hereinafter.

Turning now to FIG. 79, a generic protocol 1000 of the apparatus 700 isshown. As illustrated therein, in a first step 1010, enrichment iscarried out by reducing platelets and plasma in a sample. In embodimentswhere Dynabeads are utilized as magnetic isolation beads, a washing step1012 to remove the residuals in the Dynabead suspension may then becarried out. After enrichment, the cells are then transferred to theprocess bag 932, at step 1014. In some embodiments, a portion of theenriched cells may be stored in a first storage bag 938, at step 1016,prior to transfer into the process bag 932. At step 1018, magneticisolation beads are injected into the process bag, such as by using thesyringe 952, at step 1020. In an embodiment, the magnetic isolationbeads are Miltenyi beads or StemCell EasySep beads. Where Dynabeads areutilized, the washed Dynabeads from step 1012 are resuspended in theprocess bag 932. In an embodiment, rather than utilizing a syringe, themagnetic isolation beads may be housed in a bag or vessel that isconnected to the system, and the beads may be drawn into the system bythe pump 914.

The beads and cells in the process bag 932 are then incubated for aperiod of time, at step 1020. In embodiments where the magneticisolation beads are Miltenyi nano-sized beads, a sedimentation wash iscarried out at step 1022 to remove the excess nano-sized beads, and aportion of the incubated bead-bound cells is stored in the secondstorage bag 940, at step 1024. After incubation, the bead-bound cellsare isolated using a magnet, e.g., magnetic field plates 964, 966 ofmagnetic cell isolation holder 960, at step 1026. Residual bead-boundcells are then rinsed and isolated, at step 1028. Finally, inembodiments where Miltenyi or Dynabeads are utilized, at step 1030, theisolated bead-bound cells are collected in collection bag 950. Inembodiments where StemCell EasySep beads are utilized, the additionalstep 1032 of releasing the cells from the beads to remove the beads, andthe optional step 1034 of washing/concentrating the collected cells arecarried out.

A more detailed description of the generic protocol of FIG. 79 using theapparatus 900 is described in more detail below, with specific referenceto FIG. 80, which is a schematic illustration of the flow architecture1100 of the apparatus 900. To begin, the process of enrichment (step1010) is commenced by transferring the apheresis product containedwithin the source bag 930 and washing buffer from washing buffer bag 936to the chamber 912 for washing using washing buffer, in order to reducethe amount of platelets and serum. At this point, the enriched sourcematerial is located in the chamber 912. To begin the isolation process,a separation column received by the magnet cell isolation holder 960 isprimed by initiating a flow of a buffer from isolation buffer bag 934 tothe process bag 932 through the manifold 918 and through the column toprime the column.

As disclosed above, in certain embodiments, such as where Dynabeads areutilized as magnetic isolation beads, a washing step (step 1012) iscarried out to remove any residuals in the bead suspension buffer. Thewashing step includes injecting the beads using the syringe 952 whilecirculating in a process loop 1110 (e.g., from the process bag 932,through the peristaltic pump tubing 914, through the manifold 918, andback to the process bag 932), clearing the process loop 1110, and thencapturing the beads by flowing the process bag 932 to the isolationwaste bag 942 while the magnetic field generator 962 in ‘ON’. Inembodiments where no washing is desired, the process bag 932 is flowedto the isolation waste bag 942 to ensure that the process bag 932 isclear. As used herein, in the case of a permanent magnet, ON means thatthe magnetic retention element or material 968 (e.g., the separationcolumn, matrix or tube) is in the appropriate position within themagnetic field. OFF means that the tubing section is removed from themagnetic field.

Next, the enriched cells in the processing chamber 912 are transferredto the process bag 932 (step 1014), and an isolation buffer from theisolation buffer bag 934 is drawn into the processing chamber 912 torinse the chamber 912 of any remaining cells. After rinsing, the fluidis expelled to the process bag 932. This rinsing process may berepeated, as desired. After all of the cells have been transferred tothe process bag 932, the chamber 912 is cleaned by drawing buffer fromthe isolation buffer bag 934 into the chamber 912 and expelling thefluid to the source bag 930. This cleaning process may be repeated, asdesired.

The contents of the process bag 932 may then be mixed by circulating thecontents along the process loop 1110, before clearing the process loop1110 by returning the entire contents to the process bag 932. Asindicated above, in an embodiment, a portion of the enriched cells maybe stored at this point by transferring a portion of the contents of theprocess bag 932 to the first storage bag 938 (step 1016). The processline 1112 and first storage bag line 1114 may then be cleared.

In embodiments where the bead washing step is not utilized, beads arethen injected into the process loop 1110 using the syringe 952 and theprocess loop 1110 is cleared (step 1018). In embodiments where the beadwashing step is utilized, the beads are resuspended and circulatedthrough the process loop 1110 (step 1018) and column 968, and theprocess loop is cleared through the column 968.

As discussed above, after adding the magnetic isolation beads, the cellsmay be incubated for a period of time (step 1020). In an embodiment,prior to incubation, the contents of process bag 932 may be transferredto the second storage bag 940, and the second storage bag 940 isagitated (such as using the heating-cooling-mixing chamber 922). Thecontents of the second storage bag 940 are then transferred back to theprocess bag 932. Buffer from the isolation buffer bag 934 is then drawninto the processing chamber 912, and the chamber contents are expelledto the second storage bag 940, and then transferred to the process bag932 to rinse the second storage bag 940.

In either embodiment, the cells are then incubated along with themagnetic isolation beads by circulating the cells along the process loop1110 for a prescribed incubation time. After incubation, the processloop 1110 is cleared.

As discussed above, after incubation, the optional step of washing outexcess beads (e.g., nano-sized beads) may be carried out (step 1022).Washing out excess nano-sized beads includes initiating a flow from theprocess bag 932 to the second storage bag 940, drawing the contents ofthe second storage bag 940 into the processing chamber 912, transferringbuffer from the isolation buffer bag 934 to the process bag 932,transferring the contents of the process bag 932 to the second storagebag 940, and drawing the contents of the second storage bag 940 into theprocessing chamber. The steps of flowing from the isolation buffer bag934 to the process bag 932, and then to the second storage bag 940 maybe repeated as desired to wash out excess beads. In an embodiment, thechamber 912 may then be filled with buffer from the isolation buffer bag934, initiating rotation of the chamber 912, and then expellingsupernatant to the waste bag 742. These steps may be repeated asdesired. In an embodiment, cells in the chamber are expelled to theprocess bag 932, buffer from isolation buffer bag 934 is drawn into thechamber 932, and the chamber is then expelled to the process bag 932.This process may likewise be repeated as desired. Mixing of the processloop and clearing of the process loop are then carried out.

In some embodiments, a portion of the incubated cell population may bestored in the second storage bag 940 (step 1024). To do so, a portion ofthe contents of the process bag 932 may be transferred to the secondstorage bag 940, and then the process line and second storage line 1116are cleared.

In any of the processes described above, after incubation, thebead-bound cells are isolated using the magnets 964, 966 (step 1026).This is accomplished by flowing from the process bag 932 to the wastebag 942 while the magnetic field generator 962 is ‘ON’. Residual wasteis then cleared by pumping buffer from the isolation buffer bag 934 tothe process bag 932, and then pumping from the process bag 932 to thewaste bag 942 with the magnetic field generator 962 ‘ON’.

In an embodiment, rinsing without re-suspension may be carried out bypumping buffer from the isolation buffer bag 934 to the process bag 932,rinsing the process loop 1110, clearing the process loop 1110, andflowing from the process bag 932 to the waste bag 942 with the magneticfield generator 962 ‘ON’.

In another embodiment, rinsing via re-suspension may be carried out bypumping buffer from the isolation buffer bag 934 to the process bag 932with the magnetic field generator 962 ‘OFF’, circulating in the processloop 1110, clearing the process loop, and flowing from the process bag932 to the waste bag 942 with the magnetic field generator 962 ‘ON’.

In an embodiment, residual waste may be cleared by pumping buffer fromthe isolation buffer bag 934 to the process bag 932, and flowing fromthe process bag 932 to the waste bag 942 with the magnetic fieldgenerator 962 ‘ON’.

After rinsing and isolating the residual bead-bound cells, the isolatedbead bound cells are then collected (step 1028). Where the bead-boundcells are to be collected without releasing the cells from the beads, inone method, the media from media bag 946 is simply pumped through thecolumn 968 to the collection bag 950 with the magnetic field generator962 ‘OFF’. In another method, buffer from Isolation buffer bag 934 ispumped to the process bag 932, and the process bag 932 is then pumped tothe collection bag 950 with the magnetic field generator 962 ‘OFF’. Thissecond method provides for post-isolation washing. In a third method,media from the media bag 946 is pumped to the process bag 932 throughthe column 966 (if no post-isolation wash is needed). Alternatively,buffer from isolation buffer bag 934 is pumped to the process bag 932through the column 966 (if post-isolation wash is desired). In eitherprocess, the contents of the process bag 932 are then circulated in theprocess loop 1110, the process loop 1110 is cleared by returning to theprocess bag 932, and the contents of the process bag 932 are pumped tothe collection bag 950 to collect the bead-bound cells.

Where the bead-bound cells are to be collected after releasing the cellsfrom the beads, a number of potential processes may be carried out. Forexample, in an embodiment, the cells/beads may be resuspended with themagnet ‘OFF’ by pumping a release buffer from bag 948 through the columnto the process bag 932, circulating in the process loop 1110, and thenclearing the process loop by returning the fluid to the process bag 932.Then, incubation and collection is carried out with the magnet ‘ON’ byincubating in the process loop 1110, clearing the process loop 1110,collecting the released cells by pumping from the process bag 932through the column 966 to the collection bag 950, pumping buffer fromthe isolation buffer bag 934 to the process bag 932, and collectingresiduals by pumping the contents of the process bag 932 through thecolumn 966 to the collection bag 950. The released beads (step 1032) maythen be discarded by, with the magnet ‘OFF’, pumping buffer from theisolation buffer bag 934 through the column 966 to the process bag 932,circulating in the process loop 1110, clearing the process loop 1110,and pumping the contents of the process bag 932 to the waste bag 942.

In connection with the above, in an embodiment, washing/concentration(step 1034) may be carried out by pumping the contents of the collectionbag 950 to the processing chamber 912, pumping buffer from the isolationbuffer bag 934 to the process bag 932, and transferring the buffer fromthe process bag 932 to the processing chamber 912. Wash cycles may thenbe carried out by filling the processing chamber 912 with buffer formisolation buffer bag 934, spinning the chamber 912, expellingsupernatant to the waste bag 942, an repeating the spinning andexpelling steps as desired. Finally, transferring the cells to thecollection bag after wash/concentration may be accomplished bytransferring media from the media bag 946 to the collection bag 950,pumping the collection bag contents into the processing chamber 912,expelling the contents of the processing chamber 912 to the collectionbag 950, then manually clearing the line between the processing chamber912 and the collection bag 950.

In an embodiment, one of the bags, e.g., process bag 932 may include atop port 1118 having a filter so that sterile air may be introduced intothe system (when the process bag 932 is empty) for clearing the lines,as needed, such as in the various process steps discussed above.Clearing of the lines may be accomplished as a first step in theenrichment/isolation process and/or during the process. In anembodiment, air from the collection bag 950 may be used to clear any ofthe lines of the system (e.g., air from the collection bag 950 can beused to clear the process line 1112, then the air in the process line1112 can be used to clear the desired tubing line (i.e., line 1114,1116, etc.), thereby filling the process line 1112 with liquid from theprocess bag 932, and finally clearing the process line 1112 again usingair from the collection bag 950).

In an embodiment, the processing bag 932 be blow-molded and have a highangle on the sides (having a 3D shape with a defined air pocket aboveliquid level) to limit micron-sized beads from sticking to side walls,particularly during long promote mixing during circulation-basedincubation.

In an embodiment the syringe 952 allows for addition of small volumes(such as bead suspension aliquots) to the circulation-based flow loop1110. Moreover, fluid from the flow loop 1110 can be pulled into thesyringe 952 to further clear any residuals from the syringe 952.

In an embodiment, one of the sensors 920 may configured to measure theflow of fluid. For example, one of the sensor 920 may be a bubbledetector or an optical detector which can be used as a secondaryconfirmatory measure to ensure accurate flow control (in addition to theload cells integrated with the hooks 926. This can be used in practiceduring isolation where it is desired to flow the volume in the processbag through the magnet without introducing air into the column. The loadcell indicates that the process bag is close to empty within someexpected tolerance of load cell variability, and then the bubbledetector 920 identifies the trailing liquid/air interface in order tostop the flow. The sensor 920 can therefore be used by the controller toprevent the pulling of air into the loop which can generate slugs todislodge cells, or expose cells to dry environment, or by inadvertentlypulling material into the waste bags in situations where the pump is notstopped after full draining of the process bag. In an embodiment, thebubble detector 920 can therefore be used in combination with the loadcells integrated with the hooks to improve volume control accuracy,thereby reducing cells loss and/or preventing air from entering columntubing and column.

As alluded to above in an embodiment, air may be pulled into the loopfor the purposeful generation of an air slug that can used to dislodgebead-bound cells within the isolation column/tube, for collection. In anembodiment, a buffer solution may be circulated through the isolationcolumn to elute the bead-bound cells from the isolation column, eitherin place of, or in addition to, using an air slug.

In an embodiment, two or more peristaltic pump tubes with differentinside diameters connected serially can be employed, in order to enableexpanded range of flow rates for a single pump. To switch between tubes,the pump cover is opened, the existing tube physically removed, thedesired tube physically inserted, and the pump head is then closed.

In some embodiments, the system 900 can be used for elution ofisolated/captured bead-cell complexes. In particular, it is contemplatedthat an air-liquid interface can be used to aid in the removal ofcomplexes from tube sidewalls or column interstitial spaces. Air can becirculated through or shuffled back-and-forth through the column/tube.Without the air/liquid interface, a packed bed of beads/bead-bound cellscan be difficult to remove with flow rate control alone, withoutsignificantly increasing shear rate (which has a potential negativeimpact on cell viability). Coupled with flow rate, it is thereforepossible to remove bead-cell complexes without removing from the magnet.

In connection with the above, the system 900 supports the concept ofeluting the positively selected bead-cell complexes directly into mediaof choice (based on downstream steps). This eliminates a bufferexchange/washing step. In an embodiment, it is also envisioned to elutedirectly into media and the viral vector to start incubation. Thisconcept can also enable adding viral vector to the final bag. In anembodiment, instead of eluting bead-bound cells with buffer, media maybe used as the elution fluid. Similarly, release buffer can be used toelute StemCell beads for subsequent cell release from beads. Byreplacing buffer in portions of the system 900 with media, dilution canbe minimized.

As disclosed above, the apparatus 900 of the first module 100 is asingle kit that provides for platelet- and plasma-reduced enrichmentfollowed by magnetic isolation of target cells. The apparatus 900 isautomated so as to allow the enrichment, isolation and collection steps,and all intervening steps, to be carried out with minimal humanintervention. Like the second module 200, the first module 100 andapparatus 900 thereof is functional closed to minimize the risk ofcontamination, and is flexible so as to handle various therapyvolumes/dosages/cell concentrations, and is able to support multiplecell types in addition to CAR-T cells.

It is to be understood that the system of the present invention mayinclude the necessary electronics, software, memory, storage, databases,firmware, logic/state machines, microprocessors, communication links,displays or other visual or audio user interfaces, printing devices, andany other input/output interfaces to perform the functions describedherein and/or to achieve the results described herein. For example, thesystem may include at least one processor and system memory/data storagestructures, which may include random access memory (RAM) and read-onlymemory (ROM). The at least one processor of the system may include oneor more conventional microprocessors and one or more supplementaryco-processors such as math co-processors or the like. The data storagestructures discussed herein may include an appropriate combination ofmagnetic, optical and/or semiconductor memory, and may include, forexample, RAM, ROM, flash drive, an optical disc such as a compact discand/or a hard disk or drive.

Additionally, a software application that adapts the controller(s),e.g., controller 110, 210 and/or 310, to perform the methods disclosedherein may be read into a main memory of the at least one processor froma computer-readable medium. The term “computer-readable medium”, as usedherein, refers to any medium that provides or participates in providinginstructions to the at least one processor of the system (or any otherprocessor of a device described herein) for execution. Such a medium maytake many forms, including but not limited to, non-volatile media andvolatile media. Non-volatile media include, for example, optical,magnetic, or opto-magnetic disks, such as memory. Volatile media includedynamic random access memory (DRAM), which typically constitutes themain memory. Common forms of computer-readable media include, forexample, a floppy disk, a flexible disk, hard disk, magnetic tape, anyother magnetic medium, a CD-ROM, DVD, any other optical medium, a RAM, aPROM, an EPROM or EEPROM (electronically erasable programmable read-onlymemory), a FLASH-EEPROM, any other memory chip or cartridge, or anyother medium from which a computer can read.

While in embodiments, the execution of sequences of instructions in thesoftware application causes at least one processor to perform themethods/processes described herein, hard-wired circuitry may be used inplace of, or in combination with, software instructions forimplementation of the methods/processes of the present invention.Therefore, embodiments of the present invention are not limited to anyspecific combination of hardware and/or software. Moreover, it isenvisioned that all methods, protocols and workflows described hereincan be carried out via software, which software may a single or multipleapplications, programs, etc.

Furthermore, it is contemplated that the software may be configured tocarry out the methods, protocols and/or workflows in a fully autonomousmode, a semi-autonomous mode, or in a gated manner. In a fullyautonomous mode, the software includes instructions configured to adaptthe controller(s) of the system to run substantially an entireoperation, method, protocol or workflow from start to finishautomatically once initiated by a user or operator (i.e., withoutintervention by an operator and without requiring human touchpoints). Ina semi-autonomous mode of operation, the software includes instructionsconfigured to adapt the controller(s) of the system to run substantiallyan entire operation method, protocol or workflow from start to finishonce initiated by a user or operator, except that the software mayinstruct the controller(s) to pause operation of the bioprocessingsystem or components thereof and prompt a user or operator to takecertain specific actions necessary to carry out the operation method,protocol or workflow, such as connecting or disconnecting collection,waste, media, cell, or other bags or reservoirs, to take a sample, etc.In a gated mode of operation, the software includes instructionsconfigured to adapt the controller(s) of the system to generate a seriesof prompts directing a user or operator to take certain specific actionsnecessary to carry out a given operation method, protocol or workflowsuch as connecting or disconnecting collection, waste, media, cell, orother bags or reservoirs, to take a sample, etc., and to autonomouslycontrol system operation between each discrete operator intervention. Inthe gated mode of operation, the bioprocessing system is much moreheavily operator dependent, whereby the controller(s) only carry outpreprogrammed bioprocessing steps once initiated by an operator.

As used herein, an element or step recited in the singular and proceededwith the word “a” or “an” should be understood as not excluding pluralof said elements or steps, unless such exclusion is explicitly stated.Furthermore, references to “one embodiment” of the present invention arenot intended to be interpreted as excluding the existence of additionalembodiments that also incorporate the recited features. Moreover, unlessexplicitly stated to the contrary, embodiments “comprising,”“including,” or “having” an element or a plurality of elements having aparticular property may include additional such elements not having thatproperty.

This written description uses examples to disclose several embodimentsof the invention, including the best mode, and also to enable one ofordinary skill in the art to practice the embodiments of invention,including making and using any devices or systems and performing anyincorporated methods. The patentable scope of the invention is definedby the claims, and may include other examples that occur to one ofordinary skill in the art. Such other examples are intended to be withinthe scope of the claims if they have structural elements that do notdiffer from the literal language of the claims, or if they includeequivalent structural elements with insubstantial differences from theliteral languages of the claims.

1-15. (canceled)
 16. A system for bioprocessing, comprising: a trayhaving a plurality of sidewalls and a bottom surface defining aninterior compartment, and a generally open top, the tray beingconfigured to receive a bioreactor vessel; a pump assembly positionedadjacent to the rear sidewall of the tray; a pinch valve arraypositioned adjacent to the rear sidewall of the tray; and a tubingmodule positioned at a rear of the tray, the tubing module including: afirst tubing holder block configured to receive at least one pump tubeand hold the at least one pump tube in position for selective engagementwith the pump assembly; and a second tubing holder block configured toreceive a plurality of pinch valve tubes and hold each pinch valve tubeof the plurality of pinch valve tubes in position for selectiveengagement with a respective actuator of the pinch valve array.
 17. Thesystem of claim 16, wherein: the tubing module is integral with thetray.
 18. The system of claim 16, wherein: the tubing module isremovably received by an opening formed in a rear wall of the tray. 19.The system of claim 16, further comprising: a temperature controlledprocessing chamber having a plurality of sidewalls, a bottom surface,and a generally open top, wherein the tray is receivable by theprocessing chamber; wherein the processing chamber includes a shoe thatextends upwardly from the processing chamber and is received through afirst clearance opening in the tubing module; and wherein the at leastone pump tube is received intermediate the pump assembly and the shoe.20. The system of claim 19, wherein: the processing chamber includes ananvil that extends upwardly from the processing chamber and is receivedthrough a second clearance opening in the tubing module; and wherein theplurality of pinch valve tubes are received intermediate the pinch valvearray and the anvil.
 21. The system of claim 20, wherein: the secondtubing holder block of the tubing module includes a back plate having aplurality of apertures corresponding to the plurality of pinch valvetubes; wherein the plurality of pinch valve tubes are positionedadjacent to the back plate wall when the plurality of tubes are receivedby the second tubing holder block.
 22. The system of claim 21, wherein:the plurality of apertures includes a first column of apertures and asecond column of apertures; wherein the apertures in the first column ofapertures are offset in a vertical direction with respect to theapertures on the second column of apertures so that the apertures in thefirst column of apertures are not in horizontal alignment with theapertures in the second column of apertures.
 23. The system of claim 22,wherein: each aperture of the plurality of apertures is arranged so asto be aligned with an actuator of the pinch valve array so as to allowengagement of the actuator with the plurality of pinch valve tubesthrough the plurality of apertures.
 24. The system of claim 16, wherein:the tubing module includes at least one of a hollow fiber filter and/ora tangential flow filtration device coupled to the tubing module. 25.The system of claim 16, wherein: the tubing module includes at least onevessel tubing clip for retaining one of a drain line and a feed lineconnected to the bioreactor vessel.
 26. The system of claim 16, wherein:the first tubing holder block is configured to hold the at least onepump tube in a substantially linear orientation.
 27. The system of claim16, wherein: the tubing module includes a media line tubing holderhaving a plurality of slots configured to receive and retain mediatubing.
 28. The system of claim 16, wherein: the tubing module is one of3D printed or injection molded; and the tray is one of thermoformed orinjection molded.